Specific Cysteines in β3 Are Involved in Disulfide Bond Exchange-dependent and -independent Activation of αIIbβ3
2008; Elsevier BV; Volume: 283; Issue: 28 Linguagem: Inglês
10.1074/jbc.m802399200
ISSN1083-351X
AutoresRonit Mor-Cohen, Nurit Rosenberg, Meytal Landau, Judith Lahav, Uri Seligsohn,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoDisulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of β3 was suggested to be involved in activation of αIIbβ3. To investigate the role of specific β3 cysteines in αIIbβ3 expression and activation, we expressed in baby hamster kidney cells normal αIIb with normal β3 or β3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and β-tail domains and assessed αIIbβ3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of αIIbβ3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active αIIbβ3, implying that these bonds stabilize the inactive αIIbβ3 conformer. Mutants of the Cys-567–Cys-581 bond in EGF-4 were inactive even after exposure to αIIbβ3-activating antibodies, indicating that this bond is necessary for activating αIIbβ3. Disrupting Cys-560–Cys-583 in the EGF-3/EGF-4 or Cys-608–Cys-655 in β-tail domain resulted in αIIbβ3 activation only when Cys-560 or Cys-655 of each pair was mutated but not when their partners (Cys-583, Cys-608) or both cysteines were mutated, suggesting that free sulfhydryls of Cys-583 and Cys-608 participate in αIIbβ3 activation by a disulfide bond exchange-dependent mechanism. The free sulfhydryl blocker dithiobisnitrobenzoic acid inhibited 70% of anti-LIBS6 antibody-induced activation of wild-type αIIbβ3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in αIIbβ3 activation. These data suggest that different disulfide bonds in β3 EGF and β-tail domains play variable structural and regulatory roles in αIIbβ3. Disulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of β3 was suggested to be involved in activation of αIIbβ3. To investigate the role of specific β3 cysteines in αIIbβ3 expression and activation, we expressed in baby hamster kidney cells normal αIIb with normal β3 or β3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and β-tail domains and assessed αIIbβ3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of αIIbβ3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active αIIbβ3, implying that these bonds stabilize the inactive αIIbβ3 conformer. Mutants of the Cys-567–Cys-581 bond in EGF-4 were inactive even after exposure to αIIbβ3-activating antibodies, indicating that this bond is necessary for activating αIIbβ3. Disrupting Cys-560–Cys-583 in the EGF-3/EGF-4 or Cys-608–Cys-655 in β-tail domain resulted in αIIbβ3 activation only when Cys-560 or Cys-655 of each pair was mutated but not when their partners (Cys-583, Cys-608) or both cysteines were mutated, suggesting that free sulfhydryls of Cys-583 and Cys-608 participate in αIIbβ3 activation by a disulfide bond exchange-dependent mechanism. The free sulfhydryl blocker dithiobisnitrobenzoic acid inhibited 70% of anti-LIBS6 antibody-induced activation of wild-type αIIbβ3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in αIIbβ3 activation. These data suggest that different disulfide bonds in β3 EGF and β-tail domains play variable structural and regulatory roles in αIIbβ3. Integrin αIIbβ3 mediates platelet aggregation by serving as a receptor for fibrinogen and von Willebrand factor. Like other integrins, the affinity of αIIbβ3 for its ligands is tightly regulated by cellular events (inside-out signaling). αIIbβ3 is inactive in resting platelets, but following activation by inside-out signals it undergoes conformational changes resulting in ligand binding to its large globular head, which further modifies the conformation leading to clustering of the αIIbβ3 receptors, tyrosine phosphorylation, and cytoskeleton rearrangement (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6849) Google Scholar, 2Hughes P.E. Pfaff M. Trends Cell Biol. 1998; 8: 359-364Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 3Phillips D.R. Charo I.F. Scarborough R.M. Cell. 1991; 65: 359-362Abstract Full Text PDF PubMed Scopus (481) Google Scholar, 4Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Crossref PubMed Google Scholar, 5Calvete J.J. Proc. Soc. Exp. Biol. 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Nature. 2004; 432: 59-67Crossref PubMed Scopus (676) Google Scholar). Physiologic agonist-induced insideout signaling involves separation of the cytoplasmic tails of αIIb andβ3 that results in rearrangement of the extracellular domain of the integrin, culminating in ligand binding (10Hughes P.E. Diaz-Gonzalez F. Leong L. Wu C. McDonald J.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1996; 271: 6571-6574Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 11Calderwood D.A. Zent R. Grant R. Rees D.J. Hynes R.O. Ginsberg M.H. J. Biol. Chem. 1999; 274: 28071-28074Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). Activation of αIIbβ3 can also be induced directly by antibodies to ligand-induced binding sites (LIBS) 2The abbreviations used are: LIBS, ligand-induced binding site; BHK, baby hamster kidney; DTNB, dithiobisnitrobenzoic acid; EGF, epidermal growth factor; FITC, fluorescein isothiocyanate; WT, wild type; βTD, β-tail domain. 2The abbreviations used are: LIBS, ligand-induced binding site; BHK, baby hamster kidney; DTNB, dithiobisnitrobenzoic acid; EGF, epidermal growth factor; FITC, fluorescein isothiocyanate; WT, wild type; βTD, β-tail domain. without inside-out signaling (12Frelinger III, A.L. Du X.P. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1991; 266: 17106-17111Abstract Full Text PDF PubMed Google Scholar, 13Du 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). Recent studies suggest that exofacial disulfide exchange is involved in the conformational changes that follow αIIbβ3 activation. Both αIIb and β3 subunits contain highly conserved cysteine residues that form disulfide bonds. β3 contains 56 cysteines, of which 31 are located in four epidermal growth factor (EGF)-like domains and 8 are located in the carboxyl-terminal β-tail domain (βTD) (7Xiong 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 (1107) Google Scholar). Several naturally occurring cysteine substitutions in the EGF-like domains of β3 (C549R, C560F, C560R, and C598Y) that cause Glanzmann thrombasthenia, a severe bleeding disorder, as well as several artificial cysteine substitutions exhibit constitutively active αIIbβ3 (14Mor-Cohen R. Rosenberg N. Peretz H. Landau M. Coller B.S. Awidi A. Seligsohn U. Thromb. Haemostasis. 2007; 98: 1257-1265Crossref PubMed Scopus (37) Google Scholar, 15Ambo H. Kamata T. Handa M. Taki M. Kuwajima M. Kawai Y. Oda A. Murata M. Takada Y. Watanabe K. Ikeda Y. Biochem. Biophys. Res. Commun. 1998; 251: 763-768Crossref PubMed Scopus (40) Google Scholar, 16Ruiz C. Liu C.Y. Sun Q.H. Sigaud-Fiks M. Fressinaud E. Muller J.Y. Nurden P. Nurden A.T. Newman P.J. Valentin N. Blood. 2001; 98: 2432-2441Crossref PubMed Scopus (104) Google Scholar, 17Chen P. Melchior C. Brons N.H. Schlegel N. Caen J. Kieffer N. J. Biol. Chem. 2001; 276: 38628-38635Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 18Kamata T. Ambo H. Puzon-McLaughlin W. Tieu K.K. Handa M. Ikeda Y. Takada Y. Biochem. J. 2004; 378: 1079-1082Crossref PubMed Scopus (60) Google Scholar). All cysteines in β3 had been assumed to be disulfide bonded (19Calvete J.J. Henschen A. Gonzalez-Rodriguez J. Biochem. J. 1991; 274: 63-71Crossref PubMed Scopus (158) Google Scholar), but a recent report suggested that some exist as free sulfhydryls exhibiting properties of a redox site directly involved in integrin activation (20Yan B. Smith J.W. J. Biol. Chem. 2000; 275: 39964-39972Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Moreover, the reducing agent dithiothreitol was shown to activate αIIbβ3 and cause slow progressive platelet aggregation by a mechanism involving both disulfide bond reduction and disulfide bond exchange (21Yan B. Smith J.W. Biochemistry. 2001; 40: 8861-8867Crossref PubMed Scopus (115) Google Scholar). Two other reports by one of us showed that extracellular free thiols and enzymatically catalyzed disulfide bond exchanges are required for acquisition of a ligand binding conformation of αIIbβ3 (22Lahav J. Jurk K. Hess O. Barnes M.J. Farndale R.W. Luboshitz J. Kehrel B.E. Blood. 2002; 100: 2472-2478Crossref PubMed Scopus (167) Google Scholar, 23Lahav J. Gofer-Dadosh N. Luboshitz J. Hess O. Shaklai M. FEBS Lett. 2000; 475: 89-92Crossref PubMed Scopus (129) Google Scholar). In this context it is notable that αIIbβ3 harbors an endogenous thiol isomerase activity (24O'Neill S. Robinson A. Deering A. Ryan M. Fitzgerald D.J. Moran N. J. Biol. Chem. 2000; 275: 36984-36990Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In the present study we analyzed the role of specific disulfide bonds in the exofacial domain in promoting αIIbβ3 activation. We created mutations in cysteine residues that disrupted nine disulfide bonds in the EGF and β-tail domains of β3 and expressed them in baby hamster kidney (BHK) cells. It will be shown that different cysteines play different roles in the activation of αIIbβ3 involving disulfide bond exchange-dependent or -independent mechanisms. Materials—Dulbecco modified Eagle's medium, l-glutamine, and fetal calf serum were from Biological Industries (Beit-Haemek, Israel). Lipofectamine reagent and G418 were from Invitrogen. Hygromycin was from Roche Applied Science. Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody P2 against αIIbβ3 complex was obtained from Immunotech (Marseille, France). FITC-conjugated fibrinogen mimetic murine monoclonal antibody PAC-1 was obtained from BD Biosciences. The αIIbβ3-activating monoclonal antibody anti-LIBS6 was a gift from Dr. Mark Ginsberg (Dept. of Medicine, University of California, San Diego, La Jolla, CA). The activating monoclonal antibody PT25-2 was from Takara Bio Inc. (Shiga, Japan). The membrane impermeant-free sulfhydryl blocker dithiobisnitrobenzoic acid (DTNB) was from Sigma. Construction of Expression Vectors for Mutant cDNAs—cDNAs of αIIb or β3 in pcDNA3 vector were gifts from Dr. Peter Newman from the Blood Center of Wisconsin, Milwaukee. cDNA of αIIb was subcloned to the PvuII site of pCEP4 mammalian expression vector carrying the hygromycin resistance gene as a selection marker (Invitrogen) as previously described (25Yatuv R. Rosenberg N. Zivelin A. Peretz H. Dardik R. Trakhtenbrot L. Seligsohn U. Blood. 2001; 98: 1063-1069Crossref PubMed Scopus (21) Google Scholar). Substitutions of selected cysteine residues by serine or other residues were created in the pcDNA3/β3 vector using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using two overlapping oligonucleotide primers containing single base pair substitutions (available upon request). Correct incorporation of the mutations into the pcDNA3/β3 vectors was verified by DNA sequencing. For creation of double mutants containing two cysteine substitutions together, we first introduced one mutation into normal pcDNA3/β3 vector and then used the mutant pcDNA3/β3 clone as a template for introducing the second mutation. Co-transfection of αIIb and β3 cDNAs—BHK cells were grown in Dulbecco's modified Eagle's medium supplemented by 2 mg/ml l-glutamine and 5% fetal calf serum. The cells were co-transfected with 1 μg of normal or mutated forms of pcDNA3/β3 and 1 μg of normal pCEP4/αIIb using Lipofectamine reagent. The transfected cells were grown in a selection medium containing 0.7 mg/ml G418 and 0.5 mg/ml hygromycin. Mock cells were transfected with both pCEP4 and pcDNA3 and were selected in the same medium. At least two different transfections for each mutant or wild-type (WT) construct were performed and used for flow cytometry. Flow Cytometry of Transfected BHK Cells—Transfected BHK cells were harvested with phosphate-buffered saline supplemented with 1 mm EDTA, pelleted, and incubated in Dulbecco's modified Eagle's medium for 30 min at room temperature. Cells were pelleted again, resuspended in phosphate-buffered saline supplemented with 1 mm MgCl2 and 1 mm CaCl2 (5 × 105 cells/100 μl), and incubated for 30 min at room temperature with either 20 μl of FITC-conjugated P2 antibody or 20 μl of FITC-conjugated PAC-1 antibody. The cells were diluted to 5 × 105 cells/ml and analyzed for surface fluorescence by flow cytometry (Coulter, EPICS, Luton, UK). To measure ligand binding after activation of αIIbβ3 by anti-LIBS6 or PT25-2 antibodies, FITC-conjugated PAC-1 was added to cells suspended in phosphate-buffered saline supplemented with 0.25 mm MnCl2 (5 × 105 cells/100 μl) in the presence of 1 μl of anti-LIBS6 or 1 μg of PT25-2. These experiments were repeated in the presence of 2.5 mm DTNB, which inhibited free sulfhydryls. The base line for nonspecific binding of the antibodies was measured in mock cells. PAC-1 binding to αIIbβ3 was expressed as percent of binding obtained with P2. The effects of anti-LIBS6 and DTNB were compared by two-tailed, paired t test analysis. Alignment of EGF Domains and a Model of the EGF-3 Domain of β3—An initial alignment of 2125 EGF-like domains was taken from the Pfam data base (26Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L. Studholme D.J. Yeats C. Eddy S.R. Nucleic Acids Res. 2004; 32: D138-D141Crossref PubMed Google Scholar) (accession number PF07974). Sequences derived from a whole genome shotgun were discarded, as were sequences of variant and mutant proteins. The remaining data set of 1462 sequences was used to generate a hidden Markov model (27Eddy S.R. Curr. Opin. Struct. Biol. 1996; 6: 361-365Crossref PubMed Scopus (848) Google Scholar) subsequently utilized to collect and align 1856 EGF domain sequences from the annotated SwissProt data base (28Bairoch A. Apweiler R. Nucleic Acids Res. 1999; 27: 49-54Crossref PubMed Scopus (463) Google Scholar). From this alignment, a smaller multiple sequence alignment of representative sequences was obtained, including the EGF-3 and EGF-4 domains of human integrin β3 and additional human EGF domains for which the three-dimensional structure is available. A model of the EGF-3 domain of β3 was constructed using the program NEST with default parameters (29Petrey D. Xiang Z. Tang C.L. Xie L. Gimpelev M. Mitros T. Soto C.S. Goldsmith-Fischman S. Kernytsky A. Schlessinger A. Koh I.Y. Alexov E. Honig B. Proteins. 2003; 53: 430-435Crossref PubMed Scopus (272) Google Scholar) based on the NMR structure of the EGF-3 domain in the β2 subunit (Protein Data Bank code 1L3Y) (30Beglova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (259) Google Scholar). Effect of β3 Cysteine Substitutions on the Expression and Activation State of αIIbβ3—Fig. 1A displays the nine disulfide bonds in EGF-3, EGF-4, and β-tail domains that were disrupted. The pairing of cysteines was derived from the αvβ3 crystal structure (7Xiong 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 (1107) Google Scholar). Because Cys-544 was proposed to be bonded with Cys-523 rather than Cys-536 (30Beglova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (259) Google Scholar, 31Takagi J. Beglova N. Yalamanchili P. Blacklo S.C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11175-11180Crossref PubMed Scopus (54) Google Scholar), we also mutated the cysteine residues of the Cys-523–Cys-544 alternative pair (Fig. 1B). In most instances, we mutated each one of the cysteines of the pairs as well as both cysteines (Table 1). A total of 27 mutations were created, most with cysteine to serine substitutions; some were replicas of natural mutations detected in Glanzmann thrombasthenia patients (C549R, C560R, C560F) (14Mor-Cohen R. Rosenberg N. Peretz H. Landau M. Coller B.S. Awidi A. Seligsohn U. Thromb. Haemostasis. 2007; 98: 1257-1265Crossref PubMed Scopus (37) Google Scholar, 15Ambo H. Kamata T. Handa M. Taki M. Kuwajima M. Kawai Y. Oda A. Murata M. Takada Y. Watanabe K. Ikeda Y. Biochem. Biophys. Res. Commun. 1998; 251: 763-768Crossref PubMed Scopus (40) Google Scholar, 16Ruiz C. Liu C.Y. Sun Q.H. Sigaud-Fiks M. Fressinaud E. Muller J.Y. Nurden P. Nurden A.T. Newman P.J. Valentin N. Blood. 2001; 98: 2432-2441Crossref PubMed Scopus (104) Google Scholar). All mutants were co-expressed with normal αIIb in BHK cells and analyzed by flow cytometry. The extent of surface expression of all αIIbβ3 mutants was 17–100% of WT, except for C575S and C586S mutants, which expressed <10% of WT and hindered us from analyzing their activation state (Fig. 2A). In all but one pair (Cys-536/Cys-544), the double mutants were expressed better than the corresponding single mutants, suggesting that hydrogen bonds between the substituting serine residues stabilized the αIIbβ3 structure, compensating for the loss of disulfide bonds. The natural C549R mutation caused reduced surface expression of αIIbβ3 similarly to the corresponding C549S artificial mutation, suggesting that the disruption of the Cys-549–Cys-558 bond and not the bulky Arg residue present in the natural mutation caused the reduced expression. In contrast, the natural C560R and C560F mutations caused αIIbβ3 surface expression that was lower than the corresponding artificial C560S mutation, suggesting that the bulky substituting residues (Arg and Phe) interfered with the expression of αIIbβ3.TABLE 1Natural and artificial mutations created for disruption of nine disulfide bonds in β3DomainDisulfide bondMutations createdaSubstitutions identified in patients with Glanzmann thrombasthenia are depicted in bold letters.EGF-3Cys-536—Cys-544bDisplayed in the αvβ3 crystal structure (7). or Cys-523—Cys-544cProposed by Springer and coworkers (30, 31).C536S, C544S, C523S, C536S/C544S, C523S/C544SCys-549—Cys-558C549S, C549R, C558S, C558S/C549SEGF-3/EGF-4Cys-560—Cys583C560S, C560R, C560F, C560S/C583SEGF-4Cys-567—Cys-581C567S, C581S, C567S/C581SCys-575—Cys-586C575S, C586S, C575S/C586SCys-588—Cys-598C588S, C598S, C588S/C598SβTDCys-608—Cys-655C608S, C655S, C608S/C655SCys-614—Cys-635C614SCys-617—Cys-631C617Sa Substitutions identified in patients with Glanzmann thrombasthenia are depicted in bold letters.b Displayed in the αvβ3 crystal structure (7Xiong 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 (1107) Google Scholar).c Proposed by Springer and coworkers (30Beglova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (259) Google Scholar, 31Takagi J. Beglova N. Yalamanchili P. Blacklo S.C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11175-11180Crossref PubMed Scopus (54) Google Scholar). Open table in a new tab FIGURE 2Surface expression and activation state of αIIbβ3 complexes in cells expressing normal or mutated β3. A, surface expression of αIIbβ3 in BHK cells harboring WT αIIbβ3 or mutated β3 measured by FITC-conjugated P2 antibody. The disulfide bonds that were disrupted by the mutations are depicted above the bars. Double mutants are depicted in dotted bars. The broken line represents 50% of P2 binding to WT cells. Error bars represent mean ± S.E. of at least three experiments. B, activation state of αIIbβ3 in cells harboring WT or β3 mutants assessed by flow cytometry with FITC-conjugated PAC-1 antibody. PAC-1 binding is presented as percent of αIIbβ3 expression measured by P2. Error bars represent mean ± S.E. of at least three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The activation state of mutant αIIbβ3 complexes was determined by binding of the fibrinogen mimetic antibody PAC-1. Cells harboring disulfide bond disruptions in EGF-3 displayed a pronounced increase in PAC-1 binding. Compared with WT cells, there was a 25–33-fold increase for Cys-536–Cys-544 or Cys-523–Cys-544 bonds and a 17–24-fold increase for the Cys-549–Cys-558 bond (Fig. 2B)(p < 0.001). The effect of disruptions of the Cys-560–Cys-583 bond located at the interface between EGF-3 and EGF-4 depended on the cysteine that was mutated (Fig. 2B). Although all substitutions of Cys-560 gave rise to a 22–24-fold increase in PAC-1 binding compared with WT cells (p < 0.001), a mutation in the partner cysteine C583S did not exhibit a significant increase in binding, and a double mutant (C560S/C583S) only yielded an 8-fold increased binding (p < 0.05). Disruption of two disulfide bonds in EGF-4 domain also yielded variable results (Fig. 2B). Cells with the Cys-567–Cys-581 bond disruptions caused by mutations of Cys-567, Cys-581, or both displayed no significant increase in binding of PAC-1. In contrast, cells with Cys-588–Cys-598 bond disruptions displayed increased PAC-1 binding, a 10-fold increase for C588S (p < 0.05), a 23-fold increase for C598S (p < 0.001), and a 34-fold increase for C588S/C598S (p < 0.001). Disruption of the Cys-608–Cys-655 bond in the βTD by a C655S mutation displayed a 14-fold increase in binding PAC-1 (p < 0.001), whereas a mutation in its counterpart, C608S, or the double mutation C608S/C655S did not exhibit a significant increase in PAC-1 binding (Fig. 2B). Mutations of cysteines of two other disulfide bonds within βTD, C614S or C617S, yielded no significant increase in PAC-1 binding, which agrees with a previous report (32Butta N. Arias-Salgado E.G. Gonzalez-Manchon C. Ferrer M. Larrucea S. Ayuso M.S. Parrilla R. Blood. 2003; 102: 2491-2497Crossref PubMed Scopus (45) Google Scholar). Activation of αIIbβ3 Mutants by Activating Antibodies—To examine whether the constitutively active mutants reached their maximum activation state, we used two activating antibodies, anti-LIBS6 and PT25-2. As expected, anti-LIBS6 antibody caused a 50-fold increase in binding of PAC-1 to WT αIIbβ3 (Fig. 3A). The effect of anti-LIBS6 antibody on the αIIbβ3 mutants varied considerably; all mutants in EGF-3 exhibited no significant increase in PAC-1 binding (Fig. 3A), whereas mutants of the Cys-560–Cys-583 bond located at the interface between EGF-3 and EGF-4 displayed increased PAC-1 binding (Fig. 3B). The increase was inversely related to the extent of constitutive activation. In the Cys-560 mutants that were strongly activated, anti-LIBS6 antibody only exerted a 1.3–1.8-fold increase in PAC-1 binding; in the counterpart C583S mutant that was not constitutively active, PAC-1 binding increased by nearly 30-fold; and in the double C560S/C583S mutant, which was weakly constitutively active, anti-LIBS6 antibody caused a 5-fold increase in PAC-1 binding (p < 0.05). The Cys-567–Cys-581 bond mutants in EGF-4 that were constitutively inactive did not exhibit an increased PAC-1 binding induced by anti-LIBS6 antibody, whereas the constitutively active Cys-588–Cys-598 bond mutants displayed a 1.2–3-fold increase in PAC-1 binding (p < 0.05) (Fig. 3C). Anti-LIBS6 antibody also induced a further increase in PAC-1 binding to the Cys-608–Cys-655 mutants that was related to their constitutive activation state, a 2.6-fold increase for C655S, a 12-fold increase for C608S, and a 6.2-fold increase for C608S/C655S (p < 0.05) (Fig. 3D). All the above experiments were reproduced with PT25-2-activating antibody (data not shown). Because the epitope for anti-LIBS6 antibody is within residues 602–690 of β3 (13Du 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) and the epitope of PT25-2 is at the lower surface of the β-propeller of αIIb (33Tokuhira M. Handa M. Kamata T. Oda A. Katayama M. Tomiyama Y. Murata M. Kawai Y. Watanabe K. Ikeda Y. Thromb. Haemostasis. 1996; 76: 1038-1046Crossref PubMed Scopus (53) Google Scholar, 34Puzon-McLaughlin W. Kamata T. Takada Y. J. Biol. Chem. 2000; 275: 7795-7802Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), it appears that their effect on the mutants was not epitope-specific. Taken together our data show that whereas mutants involving Cys-560–Cys-583, Cys-588–Cys-598, or Cys-608–Cys-655 bonds could be further activated by activating antibodies, mutants with Cys-536/Cys-523–Cys-544 or Cys-549–Cys-558 bond disruptions could not be further activated, and mutants involving Cys-567–Cys-581 bond disruption remained non-active. Effect of the Free Sulfhydryl Blocker DTNB on the αIIbβ3 Activation State—To study the role of free sulfhydryls on the activation state of the β3 mutants, we selected disulfide bonds from each one of the four regions, EGF-3, EGF-3/EGF-4 boundary, EGF-4, and βTD and examined the effect of adding 2.5 mm DTNB on PAC-1 binding to αIIbβ3. Fig. 4 shows that DTNB caused a consistent, but not statistically significant, decrease in PAC-1 binding in all mutants examined except for C560S/C583S and C588S/C598S mutants, which exhibited a slight but statistically significant decrease (Fig. 4, B and C). Notably, the constitutively active mutants remained considerably active after DTNB exposure. Thus, blocking free sulfhydryls with DTNB in the selected αIIbβ3 mutants had no or small effect on their constitutively activation state. Inhibition of Anti-LIBS6 Antibody-induced vation induced by anti-LIBS6 antibody. As expected, anti-LIBS6 antibody-induced activation of WT αIIbβ3 measured by PAC-1 binding declined by 70% in the presence of 2.5 mm DTNB (Fig. 5) (p < 0.001). A less pronounced but statistically significant decline by 32, 32, and 35% was observed for C583S (Fig. 5A), C588S (Fig. 5B), and C608S (Fig. 5C) mutants, respectively (p < 0.005). In contrast, DTNB had a non-statistically significant inhibitory effect on anti-LIBS6 antibody-induced increase in PAC-1 binding to all other mutants examined. The EGF-3 and Cys-567–Cys-581 mutants that were not activated by anti-LIBS6 antibody were not tested with DTNB. Alignment of EGF-like Domains—Our data show that substitutions of different cysteines in the EGF and β-tail domains elicit diverse effects on the activation state of αIIbβ3. Because EGF-like domains are conserved and abundant in proteins (35Appella E. Weber I.T. Blasi F. FEBS Lett. 1998; 231: 1-4Crossref Scopus (239) Google Scholar), we performed a sequence alignment analysis to seek a potential pattern in this diversity. Ten conserved EGF-like domains are presented in Fig. 6. All EGF domains share a distinctive motif of 6 conserved cysteines (C1–C6) that typically form three internal disulfide bonds in a 1–3, 2–4, and 5–6 pattern, although C1, C2, C3, and C4 display in some instances alternative patterns of disulfide bridges that have the potential of rearranging disulfide bonds (36Barton W.A. Tzvetkova-Robev D. Miranda E.P. Kolev M.V. Rajashankar K.R. Himanen J.P. Nikolov D.B. Nat. Struct. Mol. Biol. 2006; 13: 524-532Crossref PubMed Scopus (94) Google Scholar, 37Chen V.M. Hogg P.J. J. Thromb. Haemostasis. 2006; 4: 2533-2541Crossref PubMed Scopus (79) Google Scholar). 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