Boxu Yan

Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of neurodegenerative diseases and stroke. However, the mechanism of MMP activation remains unclear. We report that MMP activation involves S-nitrosylation. During cerebral ischemia in vivo, MMP-9 colocalized with neuronal nitric oxide synthase. S-Nitrosylation activated MMP-9 in vitro and induced neuronal apoptosis. Mass spectrometry identified the active derivative of MMP-9, both in vitro and in vivo, as a stable sulfinic or sulfonic acid, whose formation was triggered by S-nitrosylation. These findings suggest a potential extracellular proteolysis pathway to neuronal cell death in which S-nitrosylation activates MMPs, and further oxidation results in a stable posttranslational modification with pathological activity.

Cellular regulation of the ligand binding affinity of integrin adhesion receptors (integrin activation) depends on the integrin beta cytoplasmic domains (tails). The head domain of talin binds to several integrin beta tails and activates integrins. This head domain contains a predicted FERM domain composed of three subdomains (F1, F2, and F3). An integrin-activating talin fragment was predicted to contain the F2 and F3 subdomains. Both isolated subdomains bound specifically to the integrin beta3 tail. However, talin F3 bound the beta3 tail with a 4-fold higher affinity than talin F2. Furthermore, expression of talin F3 (but not F2) in cells led to activation of integrin alpha(IIb)beta3. A molecular model of talin F3 indicated that it resembles a phosphotyrosine-binding (PTB) domain. PTB domains recognize peptide ligands containing beta turns, often formed by NPXY motifs. NPX(Y/F) motifs are highly conserved in integrin beta tails, and mutations that disrupt this motif interfere with both integrin activation and talin binding. Thus, integrin binding to talin resembles the interactions of PTB domains with peptide ligands. These resemblances suggest that the activation of integrins requires the presence of a beta turn at NPX(Y/F) motifs conserved in integrin beta cytoplasmic domains.

Talin links integrin beta cytoplasmic domains to the actin cytoskeleton and is involved in the clustering and activation of these receptors. To understand how talin recognizes integrin beta cytoplasmic domains, we configured surface plasmon resonance methodology to measure the interaction of talin with the beta3 integrin cytoplasmic domain. Here we report that the N-terminal approximately 47-kDa talin head domain (talin-H) has a 6-fold higher binding affinity than intact talin for the beta3 tail. The affinity difference is mainly due to a difference in k(on). Calpain cleavage of intact talin released talin-H and resulted in a 16-fold increase in apparent K(a) and a 100-fold increase in apparent k(on). The increase in talin binding after cleavage was greater than predicted for stoichiometric liberation of free talin-H. This additional increase in binding was due to cooperative binding of talin-H and talin rod domain to the beta3 tail. Talin resembles ERM (ezrin, radixin, moesin) proteins in possessing an N-terminal FERM (band four-point-one, ezrin, radixin, moesin) domain. These data show that the talin FERM domain, like that in the ERM proteins, is masked in the intact molecule. Furthermore, they suggest that talin cleavage by calpain may contribute to the effects of the protease on the clustering and activation of integrins.

Integrin adhesion receptors contain an on/off switch that regulates ligand binding affinity and cell adhesion. The switch from “off” to “on” is commonly referred to as integrin activation. The objective of this study was to gain insight into the nature of the on/off switch in platelet integrin αIIbβ3. Here, we show that a select group of the cysteines, located within the extracellular cysteine-rich domain of the β subunit, remain unpaired. These unpaired cysteine residues exhibit the properties of a redox site involved in integrin activation. Alterations to the redox site prevent the inter-conversion between resting and active integrin. Altogether, the study establishes integrin as a direct target for redox modulation, revealing an unappreciated link between cell adhesion and redox biology. Integrin adhesion receptors contain an on/off switch that regulates ligand binding affinity and cell adhesion. The switch from “off” to “on” is commonly referred to as integrin activation. The objective of this study was to gain insight into the nature of the on/off switch in platelet integrin αIIbβ3. Here, we show that a select group of the cysteines, located within the extracellular cysteine-rich domain of the β subunit, remain unpaired. These unpaired cysteine residues exhibit the properties of a redox site involved in integrin activation. Alterations to the redox site prevent the inter-conversion between resting and active integrin. Altogether, the study establishes integrin as a direct target for redox modulation, revealing an unappreciated link between cell adhesion and redox biology. activation state-1 activation state-2 polyacrylamide gel electrophoresis polyvinylidene difluoride horseradish peroxidase protein-disulfide isomerase dithiothreitol bovine serum albumin sodium nitroprusside mass spectroscopy matrix-assisted laser desorption/ionization time-of-flight N-(6-[Biotinamido])hexyl-3′-(2′-pyridyldithio)propionamide 1-biotinamido-4-[4′-(maleimidomethyl)-cyclohexane- carboxamido]butane Integrins are transmembrane receptors that mediate cell adhesion and cell migration (1Ruoslahti E. Annu. Rev. Cell Dev. Biol. 1996; 12: 697-715Crossref PubMed Scopus (2589) Google Scholar, 2Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar). The integrin protein family is directly involved in most cell-matrix contacts and cell adhesion events. Many pathologic events, including tumor progression, angiogenesis, and vascular disease, also involve integrins (3Ruoslahti E. Giancotti F.G. Cancer Cells. 1989; 1: 119-126PubMed Google Scholar, 4Varner J.A. Cheresh D.A. Curr. Opin. Cell Biol. 1996; 8: 724-730Crossref PubMed Scopus (468) Google Scholar, 5Coller B.S. Anderson K. Weisman H.F. Thromb. Haemostasis. 1995; 74: 302-308Crossref PubMed Scopus (151) Google Scholar). In most cases cell adhesion is stringently regulated, both spatially and temporally. Spatial regulation is achieved by the expression patterns of the integrins and their various ligands. Temporal regulation of adhesion is conferred by a process called integrin “activation.” Activation unshields the integrin ligand binding site, increasing its ligand binding affinity. The activation, and de-activation, of integrins is crucial to events like morphogenesis, tumor cell invasion, and platelet aggregation (6Martin-Bermudo M.D. Dunin-Borkowski O.M. Brown N.H. J. Cell Biol. 1998; 141: 1073-1081Crossref PubMed Scopus (51) Google Scholar, 7Palecek S.P. Loftus J.C. Ginsberg M.H. Lauffenburger D.A. Horwitz A.F. Nature. 1997; 385: 537-540Crossref PubMed Scopus (1194) Google Scholar, 8Marguerie G.A. Edgington T.S. Plow E.F. J. Biol. Chem. 1980; 255: 154-161Abstract Full Text PDF PubMed Google Scholar). The subject of this study is platelet integrin αIIbβ3, an excellent paradigm of the integrin protein family. Integrin αIIbβ3 is a particularly relevant model for the study of integrin activation because its function on the platelet requires physiologic activation (9Phillips D.R. Charo I.F. Scarborough R.M. Cell. 1991; 65: 359-362Abstract Full Text PDF PubMed Scopus (482) Google Scholar). Integrin αIIbβ3 is maintained in a resting state on circulating platelets. However, agonists like ADP or thrombin induce activation of the integrin. This activation facilitates the binding of soluble fibrinogen, leading to the formation of a platelet aggregate, or thrombus, that halts the loss of blood. Consequently, activation of αIIbβ3 is key for normal hemostasis. Importantly, though, improper activation of αIIbβ3 can have lethal consequences. Rupture of atherosclerotic plaques can cause activation of αIIbβ3 on platelets and can lead to myocardial infarct (10Jang Y. Lincoff A.M. Plow E.F. Topol E.J. J. Am. Coll. Cardiol. 1994; 24: 1591-1601Crossref PubMed Scopus (381) Google Scholar). The precise mechanism by which integrins are activated and de-activated is still not completely understood. A significant body of work has focused on how alterations to integrin cytoplasmic domains control activation. In one well supported model, changes in the conformation of the cytoplasmic tails are thought to release a conformational constraint, or open an “integrin hinge” (11Hughes 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 (511) Google Scholar). The release of this hinge is believed to translate into conformational rearrangements in the extracellular face of the integrin that expose the ligand binding site. The physical association of the integrin cytoplasmic domain with regulatory proteins (12Calderwood 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 (564) Google Scholar, 13Shattil S.J. O'Toole T. Eigenthaler M. Thon V. Williams M. Babior B.M. Ginsberg M.H. J. Cell Biol. 1995; 131: 807-816Crossref PubMed Scopus (167) Google Scholar), and the connection of these domains to intracellular signaling pathways (14Chen Y.P. O'Toole T.E. Shipley T. Forsyth J. LaFlamme S.E. Yamada K.M. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1994; 269: 18307-18310Abstract Full Text PDF PubMed Google Scholar), like those controlled by Ha-Ras and R-Ras (15Hughes 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, 16Zhang Z. Vuori K. Wang H.-G. Reed J.C. Ruoslahti E. Cell. 1996; 85: 61-69Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar), also strongly support the idea that activation can be controlled from the cytoplasm. Activation of integrins by events in the cytoplasm has been termed “inside-out” signaling (for reviews, see Refs. 2Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar, 17Shattil S.J. Ginsberg M.H. J. Clin. Invest. 1997; 100 Suppl. 11: S91-S95Google Scholar, and 18Dedhar S. Hannigan G.E. Curr. Opin. Cell Biol. 1996; 8: 657-669Crossref PubMed Scopus (347) Google Scholar). The physical size of the integrin extracellular domains far exceeds the size of the cytoplasmic domains, and suggests ample opportunity for regulation at the extracellular face. We recently compared the resting and active forms of αIIbβ3 by high resolution peptide mapping, and identified a difference in their conformation at a disulfide-bonded knot in the extracellular domain of β3 (19Yan B. Hu D.D. Knowles S.K. Smith J.W. J. Biol. Chem. 2000; 275: 7249-7260Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This knot comprises the cysteine-rich domain of β3 and a small segment of the amino terminus. The two are connected by a single long range disulfide bond (20Beer J. Coller B.D. J. Biol. Chem. 1989; 264: 17564-17573Abstract Full Text PDF PubMed Google Scholar, 21Calvete J.J. Henschen A. Gonzalez-Rodriguez J. Biochem. J. 1991; 274: 63-71Crossref PubMed Scopus (158) Google Scholar). No information is available to suggest a mechanism by which the conformational changes to this domain in the ectodomain are enacted during activation. Does the extracellular domain simply respond to conformational modulation initiated within the cytoplasm, or can activation be controlled at this site in the ectodomain independent of factors in the cytoplasm? Over the last decade, there has been an increased appreciation for the role of redox chemistry in the regulation of biological systems. For example, the vasoactivity of hemoglobin, the function of the NMDA receptor, and the regulation of transcription by OxyR, are all regulated by redox modulation at specific cysteine residues (22Jia L. Bonaventura C. Bonaventura J. Nature. 1996; PubMed Scopus Google Scholar, D.R. 1999; 24: Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). integrin function be in an by redox rearrangements within the cysteine-rich of this from of the on For example, can platelet events that are to involve integrin αIIbβ3 M.W. R.M. S. PubMed Scopus Google Scholar, J. J. J. J. S. A. 1992; PubMed Scopus Google Scholar, J.A. D.J. J. Thromb. PubMed Scopus Google Scholar). has been to the that redox like cell adhesion by direct on has been that redox modulation within an integrin was particularly the that all of the cysteine residues within integrins are we show that αIIbβ3 a small of cysteines, and that the redox of these residues can directly activation These the direct link between redox and cell-matrix and have for the and The two forms of integrin αIIbβ3, activation state-1 and to B. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, B. D.R. Biochem. PubMed Scopus Google Scholar, R. M.D. S. S. Ruoslahti E. PubMed Scopus Google Scholar). For affinity of αIIbβ3, the peptide was to to the the of the ligand binding affinity in which the peptide was to the with an This was we have B. L. J. Y. Biochem. J. 1992; 8: Scholar). of αIIbβ3 was with in the of was to αIIbβ3 of 100 in 100 of A was in a of to and with integrin at for The was by to the was to and at for the with A the with dithiothreitol and the on mass 1994; Google Scholar, D.J. Biol. 1997; Google was to the of the of The for this those of (19Yan B. Hu D.D. Knowles S.K. Smith J.W. J. Biol. Chem. 2000; 275: 7249-7260Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, F. Smith J.W. Biochem. 2000; PubMed Scopus Google Scholar). with a mass with a laser in was from into of with and from on an albumin as S. J. 1989; PubMed Scopus Google Scholar). aggregation with of platelets at with in an of cysteine residues was with and to the αIIbβ3 was with of these at for was by and the integrin was to as the cysteines, αIIbβ3 was with Integrin was by with and with The integrin was with and the was with The was in at and to to the of into integrin. platelets with by with and αIIbβ3 was by an affinity with The A αIIbβ3, was in This was to with control or which to β3 J.W. D.J. Cheresh D.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The with in and and on to which with and to the was by with on for by to of This was to all of the as by the of the integrin to to The of of on this activation was by with by The was with for on and to The ligand binding function of was with affinity or long was to a resting conformation by with a of sodium nitroprusside and and and to of in A. The was to for at for ligand binding function by their to to affinity in which the is to the directly or an all integrins contain a of in their extracellular domains, these are all believed to be This is on disulfide on β3 J.J. Henschen A. Gonzalez-Rodriguez J. Biochem. J. 1991; 274: 63-71Crossref PubMed Scopus (158) Google Scholar). the of that the bond of within the β3 not be because are within a single Here, we the of cysteine residues within integrin two and a to with has because can be from the by The resting and active of αIIbβ3, and a of control with of these proteins with to for of to which and protein-disulfide both of which contain and also strongly both and on the In the of with of integrin was on and the also β3 integrin. by this be by with These are the to that integrin cysteine In all the was to a and with the However, the that a small of the a small that was during to of the integrin unpaired cysteine residues This was by and with and the integrin to with an affinity both forms of the integrin be from by the affinity This that all of the integrin unpaired the that the in integrin an of we to αIIbβ3 on the platelet from on of S. J. 1989; PubMed Scopus Google and to was by and the platelets with A of and in the to that disulfide not as a of integrin αIIbβ3 was and of this with to in platelet β3 was with In that platelet β3 was with the to the on the platelet not No was in the from or in from platelets not the of the cysteine residues within αIIbβ3, the integrin was with and with the of was in the of which integrin to (19Yan B. Hu D.D. Knowles S.K. Smith J.W. J. Biol. Chem. 2000; 275: 7249-7260Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, B. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). of integrin on The was with which a bond in protein in this The at to an that is from the The was and with as The mass from two independent are and to peptide from β3 are with and mass that all of these from the and from the to from the cysteine-rich domain of The of of the within β3 is in on these we that the comprises a domain that residues of These the cysteine residues of the integrin to the disulfide-bonded knot that conformational in resting active integrin (19Yan B. Hu D.D. Knowles S.K. Smith J.W. J. Biol. Chem. 2000; 275: 7249-7260Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, S. Y. Y. R. Z. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, T. V. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). to and exhibit a difference in the of unpaired cysteine This was by of integrin with These in to expose the integrin was with and the of into the protein was by in the of was of and from to The show that between and cysteines, and that between and Importantly, in of was to have two a that to one disulfide of in in a We and have that of resting αIIbβ3 with can to an active state (19Yan B. Hu D.D. Knowles S.K. Smith J.W. J. Biol. Chem. 2000; 275: 7249-7260Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, B. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, Thromb. Haemostasis. 1995; PubMed Scopus (51) Google Scholar). This activation from the of a single disulfide or from bond between the and In an to between these we the of the within on activation. Activation of the integrin was by its to to two affinity The binding site within the resting of αIIbβ3 is and this can to affinity the peptide is from the by a long In activated αIIbβ3 has a binding site and can to affinity the peptide is directly to the (19Yan B. Hu D.D. Knowles S.K. Smith J.W. J. Biol. Chem. 2000; 275: 7249-7260Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, B. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, Coller B.S. 1992; PubMed Google Scholar). in is to to activated with and of the within with its activation by with the activation of of the as of the be to to activation with The of not to an of the binding site on the integrin by because is still to to in which the peptide is to the a long of the cysteine residues within by on its binding function These show that of the in can prevent its activation directly its binding site. These strongly that disulfide bond is involved in the activation This was to of the cysteine residues of αIIbβ3 on the platelet prevent activation. In this we platelet aggregation as a relevant of activation of αIIbβ3. from the of and the cysteine residues on the platelet by a with as and In the of was as an have Thromb. Haemostasis. 1995; PubMed Scopus (51) Google Scholar, Thromb. Haemostasis. PubMed Scopus Google Scholar), aggregation of platelets the of aggregation and the size of the was platelets are by physiologic of platelets with to of the platelets to aggregation with a physiologic platelet of platelets with also aggregation of platelets We the that also on platelet and that these be involved in the activation the between the with and on the platelet strongly a link between the cysteine residues in activation. A of to the that of the within to a resting integrin. and exhibit we that of to be to to we to and its activation state by its to affinity with and long of direct with nitroprusside as and with a of nitroprusside and in of to nitroprusside on the integrins to to a In of during to the the of the integrin to to the is to that the of by is not simply of the integrin because its ligand binding function is is still to to in which the peptide is from the by a long a conformational that the binding site. In a of we the of to the de-activated was de-activated with to redox and with in and with the a de-activated by to and is to the However, this de-activated integrin be by as by its to the affinity not this with also to the migration of on This also disulfide and to during of these to of to the of cysteine residues in integrin. by the of into the protein as an of cysteines, with this to These show that the of and the cysteine residues within The de-activated that was with on cysteines, that the by can be by The that a of and are to to a resting state suggests that the leading to be by A. Biol. 1998; PubMed Scopus Google Scholar), and a of within a protein redox site. These and of cysteine residues on the or disulfide bond or at this we the involved in these the of this study a link between the of within the redox site, and the activation state of the integrin. also show that physiologic redox that are to on platelets in can the integrins activation state in cysteine residues are a of all integrin β on disulfide has been that all of these residues are in disulfide J.J. Henschen A. Gonzalez-Rodriguez J. Biochem. J. 1991; 274: 63-71Crossref PubMed Scopus (158) Google Scholar). No of unpaired within an integrin has been In the we cysteine residues within the These cysteine residues are on integrin and on the platelet We have been to the to a domain of residues that the cysteine-rich domain of the β3 This is connected to a cysteine-rich in the of the protein by a long range disulfide We these domains the redox site and as key domains in the process of activation. In with in the the of the study can be into that support the idea that the in β3 a redox site with a role in integrin activation. and have a of cysteine that an of one disulfide bond with a in activation This is also with a in the of on the platelet platelet C. J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). of the the activation of both in and on the platelet These are with a disulfide bond mechanism like that in in a process of disulfide bond activation. These are to that αIIbβ3 on platelets Thromb. Haemostasis. 1995; PubMed Scopus (51) Google Scholar), on B.S. 1995; PubMed Google Scholar, B.S. F. J. Biol. 1998; PubMed Scopus Google Scholar), and on the cell A. J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). activation is not to all of the involved in physiologic activation, the that aggregation suggests that bond is also an in physiologic activation. the idea for a redox site involved in activation is by the that can be to a resting state by the of and The that this de-activated can be by the that these are on a regulatory redox site. These are also with the of M.W. R.M. S. PubMed Scopus Google Scholar), and of J. J. J. J. S. A. 1992; PubMed Scopus Google S. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the conformational changes that with activation can be to domains within the disulfide-bonded knot of For example, the that on the activation of both β3 integrins T. V. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, T. V. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), to the residues of β3 S. Y. Y. R. Z. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). These residues are into with the cysteine-rich of β3 by the disulfide bond between and Consequently, changes at the redox site within the cysteine-rich domain are to be to conformational changes at this in the terminus. The that in the of the integrin also on the conformational changes that activation of that integrin A. J.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). peptide study identified key conformational between and at the disulfide-bonded knot that the redox site (19Yan B. Hu D.D. Knowles S.K. Smith J.W. J. Biol. Chem. 2000; 275: 7249-7260Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). within the cysteine-rich domain can cause activation of integrin. For example, J. E.J. B. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google that a the domain of is in an activated In with of the these suggest that the cysteine-rich domain an conformational on the ligand binding site. In a within the cysteine-rich domain of β3 the integrin to be in a activated conformation on Y. S. S. M. M. Y. Y. Shattil S.J. 1999; PubMed Google Scholar). there is that the activation of αIIbβ3 on the platelet can be controlled by platelet protein-disulfide isomerase in the study the integrin redox site was a physiologic role for in the integrin redox site not be can the formation of from and also the of disulfide H.F. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). has been identified on the platelet K. M. 1995; PubMed Google Scholar), and of are to prevent activation of αIIbβ3 on the platelet M. J. 1999; PubMed Scopus Google Scholar). redox can be regulated in all with of A. Biol. 1998; PubMed Scopus Google Scholar, Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). precise be with on the of this we can at a that The of the study are most with the mechanism in In this resting and active integrins in the and of unpaired activation this site is to involve a of that conformational These are to the of integrin ligand binding by and A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the integrin conformational by Loftus and J.C. J. Clin. Invest. 1997; 100 Suppl. 11: Scholar). one also how all integrin redox also within the by and A. Biol. 1998; PubMed Scopus Google Scholar). This of the to that biological from the alterations that with We that involve small redox like or the of in the one that to a cysteine in β3 the integrin in a resting integrin activation be regulated Many of are with these two are within the cysteine-rich domain of and cysteine residues are by the thought to be key to in proteins E.J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In to the of within we two small and residues that are in the integrin by peptide one of the and cysteine the that at these the study can be an of the at the redox site. be to the that the integrin redox site, that of the involved in are and to there be one in at the redox site that has physiologic The “inside-out” signaling is to the activation of integrin factors in the cytoplasm. a of that signaling can control activation state (12Calderwood 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 (564) Google Scholar, Y.P. O'Toole T.E. Shipley T. Forsyth J. LaFlamme S.E. Yamada K.M. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1994; 269: 18307-18310Abstract Full Text PDF PubMed Google Scholar, 16Zhang Z. Vuori K. Wang H.-G. Reed J.C. Ruoslahti E. Cell. 1996; 85: 61-69Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, S.E. Yamada Yamada K.M. J. Cell Biol. 1994; PubMed Scopus Google Scholar), the of this suggest an of that integrin activation be controlled directly by a redox site in the extracellular independent of factors in the cytoplasm. The two pathways to activation, and redox modulation, The redox switch and changes in activation state that the cytoplasm. signaling a role in control of activation, a process that from by the intracellular signaling In the regulation of cell be to how or these two to activation The also at a mechanism for the of on platelet including and all platelet events J.A. D.J. J. Thromb. PubMed Scopus Google Scholar, J. J.A. J. J. Clin. Invest. PubMed Scopus Google Scholar). the platelets of exhibit increased and platelet R. K. Knowles C. J. 1999; PubMed Scopus Google Scholar). direct of platelet function in A. L. T. J. Clin. Invest. 1998; PubMed Scopus Google Scholar). These between and platelet function have well B. B. J. J.A. 1998; PubMed Scopus (151) Google Scholar). the of on platelets have been to the of to the of in the cytoplasm, on the integrin redox site suggest that the integrin redox site be a direct target for of the well between both and αIIbβ3 to and disease, also suggest that the redox site on integrin be by at This the which have not well in 2000; Full Text Full Text PDF PubMed Scopus Google Scholar).

Integrin alphaIIbbeta3 plays a pivotal role in hemostasis and thrombosis by mediating platelet adhesion and platelet aggregation. Integrin alphaIIbbeta3 contains an on/off switch that regulates its ligand binding affinity. The switch from "off" to "on" is commonly referred to as integrin activation. We recently identified a redox site within the extracellular domain of the platelet integrin alphaIIbbeta3 that exhibits many properties that one might expect of the on/off switch [Yan, B., and Smith, J. W. (2000) J. Biol. Chem. 275, 39964-39972]. Several independent reports show that reducing agents, such as dithiothreitol, can activate integrins. The objective of the present study was to determine if the effects of DTT can be attributed to a perturbation at the integrin redox site. Indeed, we find that DTT reduces two disulfide bonds within the integrin's cysteine-rich domain. Such bond reduction leads to global conformational changes within both alphaIIb and beta3 and the opening of the RGD and fibrinogen binding sites. These findings causally link the reduction of disulfide bonds within the integrin's redox site to transitions in the integrin's activation state.