Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains

Integrin alpha v beta 3, a widely distributed fibrinogen receptor, recognizes the RGD572-574 motif in the alpha chain of human fibrinogen. However, this motif is not conserved in other species, nor is it required for alpha v beta 3-mediated fibrin clot retraction, suggesting that fibrinogen may have other alpha v beta 3 binding sites. Fibrinogen has conserved C-terminal domains in its alpha (E variant), beta, and gamma chains (designated alpha EC, beta C, and gamma C, respectively), but their function in cell adhesion is not known, except that alpha IIb beta 3, a platelet fibrinogen receptor, binds to the gamma C HHLGGAKQAGDV400-411 sequence. Here we used mammalian cells expressing recombinant alpha v beta 3 to show that recombinant alpha EC and gamma C domains expressed in bacteria specifically bind to alpha v beta 3. Interaction between alpha v beta 3 and gamma C or alpha EC is blocked by LM609, a function-blocking anti-alpha v beta 3 mAb, and by RGD peptides. alpha v beta 3 does not require the HHLGGAKQAGDV400-411 sequence of gamma C for binding, and alpha EC does not have such a sequence, indicating that the alpha v beta 3 binding sites are distinct from those of alpha IIb beta 3. A small fragment of gamma C (residues 148-226) supports alpha v beta 3 adhesion, suggesting that an alpha v beta 3 binding site is located within the gamma chain 148-226 region. We have reported that the CYDMKTTC sequence of beta 3 is responsible for the ligand specificity of alpha v beta 3. gamma C and alpha EC do not bind to wild-type alpha v beta 1, but do bind to the alpha v beta 1 mutant (alpha v beta 1-3-1), in which the CYDMKTTC sequence of beta 3 is substituted for the corresponding beta 1 sequence CTSEQNC. This suggests that gamma C and alpha EC contain determinants for fibrinogen's specificity to alpha v beta 3. These results suggest that fibrinogen has potentially significant novel alpha v beta 3 binding sites in gamma C and alpha EC.     

Fibrinogen binds to integrin alpha(5)beta(1) via the carboxyl-terminal RGD site of the Aalpha-chain

Fibrinogen interactions with vascular endothelial cells are implicated in various physiological and pathophysiological events, including angiogenesis and wound healing. We have shown previously that integrin alpha(5)beta(1) is a fibrinogen receptor on endothelial cells [Suehiro, K., Gailit, J., and Plow, E.F. (1997) J. Biol. Chem. 272, 5360-5366]. In the present study, we have characterized fibrinogen interactions with purified alpha(5)beta(1) and have identified the recognition sequence in fibrinogen for alpha(5)beta(1). The binding of fibrinogen to immobilized alpha(5)beta(1) was selectively supported by Mn(2+). Fibrinogen bound to purified alpha(5)beta(1) in a time-dependent, specific, and saturable manner in the presence of Mn(2+), and the binding was blocked completely by Arg-Gly-Asp (RGD)-containing peptides and by anti-alpha(5) and anti-alpha(5)beta(1) monoclonal antibodies. A monoclonal antibody directed to the C-terminal RGD sequence at Aalpha572-574 significantly inhibited the binding of fibrinogen to alpha(5)beta(1), whereas monoclonal antibodies directed to either the N-terminal RGD sequence at Aalpha95-97 or the C-terminus of the gamma-chain did not. Furthermore, substituting RGE for RGD at position Aalpha95-97 in recombinant fibrinogen had a minimal effect on binding, whereas substituting RGE for RGD at position Aalpha572-574 decreased binding by 90%. These results demonstrate that the C-terminal RGD sequence at Aalpha572-574 is required for the interaction of fibrinogen with alpha(5)beta(1).     

A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD- containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.     

Solution structures and integrin binding activities of an RGD peptide with two isomers

The Arg-Gly-Asp (RGD) sequence serves as the primary integrin recognition site in extracellular matrix proteins, and peptides containing this sequence can mimic the activities of the matrix proteins. Depending on the context of the RGD sequence, an RGD-containing peptide may bind to all of the RGD-directed integrins, to a few, or to only a single one. We have previously isolated from a phage-displayed peptide library a cyclic peptide that binds avidly to the alpha(v)beta3 and alpha(v)beta5 integrins but does not bind to other closely related integrins. This peptide, ACDCRGDCFCG, exists in two natural configurations depending on internal disulfide bonding. The peptide with the 1-4; 2-3 disulfide bond arrangement accounts for most of the alpha(v) integrin binding activity, whereas the 1-3; 2-4 peptide is about 10-fold less potent. Solution structure analysis by nuclear magnetic resonance reveals an entirely different presentation of the RGD motif in the two isomers of RGD-4C. These results provide new insight into the ligand recognition specificity of integrins.     

Identification of a novel binding site for platelet integrins alpha IIb beta 3 (GPIIbIIIa) and alpha 5 beta 1 in the gamma C-domain of fibrinogen

The interactions of platelets with fibrinogen mediate a variety of responses including adhesion, platelet aggregation, and fibrin clot retraction. Whereas it was assumed that interactions of the platelet integrin alpha IIb beta 3 with the AGDV sequence in the gamma C-domain of fibrinogen and/or RGD sites in the A alpha chains are involved in clot retraction and adhesion, recent data demonstrated that fibrinogen lacking these sites still supported clot retraction. These findings suggested that an unknown site in fibrinogen and/or other integrins participate in clot retraction. Here we have identified a sequence within gamma C that mediates binding of fibrinogen to platelets. Synthetic peptide duplicating the 365-383 sequence in gamma C, designated P3, efficiently inhibited clot retraction in a dose-dependent manner. Furthermore, P3 supported platelet adhesion and was an effective inhibitor of platelet adhesion to fibrinogen fragments. Analysis of overlapping peptides spanning P3 and mutant recombinant gamma C-domains demonstrated that the P3 activity is contained primarily within gamma 370-383. Integrins alpha IIb beta 3 and alpha 5 beta 1 were implicated in recognition of P3, since platelet adhesion to the peptide was blocked by function-blocking monoclonal antibodies against these receptors. Direct evidence that alpha IIb beta 3 and alpha 5 beta 1 bind P3 was obtained by selective capture of these integrins from platelet lysates using a P3 affinity matrix. Thus, these data suggest that the P3 sequence in the gamma C-domain of fibrinogen defines a previously unknown recognition specificity of alpha IIb beta 3 and alpha 5 beta 1 and may function as a binding site for these integrins.     

Distinct biological consequences of integrin alpha v beta 3-mediated melanoma cell adhesion to fibrinogen and its plasmic fragments.

Fibrinogen/fibrin and its proteolytic fragments serve as potential adhesive substrates during thrombosis, wound healing, and cancer. In this report we examined the biological response of human melanoma cells exposed to fibrinogen and its naturally occurring plasmic breakdown products that are known constituents of the tumor stroma. Plasmin treatment of fibrinogen first results in fragment X, which is characterized by removal of the COOH-terminal portion of the alpha chain including an RGD sequence (A alpha 572-575). Further digestion leads to fragment D comprising primarily an intact COOH-terminal stretch of the gamma chain containing the platelet adhesion sequence HHLGGAKQAGDV. In a sensitive adhesion assay M21 human melanoma cells utilized integrin alpha v beta 3 to attach to all three of these ligands. However, only intact fibrinogen promoted significant cell spreading, while fragment X produced minimal spreading and fragment D promoted only adhesion. These results indicate that fibrinogen contains at least two alpha v beta 3-dependent adhesive sites and these promote distinct biological responses of human melanoma cells. The differential functional properties of these ligands directly correlate to their relative binding affinity for purified alpha v beta 3 as measured in a solid-phase receptor binding assay. These results provide evidence that a single integrin can promote distinct biological signals depending on the molecular nature of the ligand binding event.     

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) recognizes multiple sites in fibronectin.

The binding of fibronectin (Fn) to several integrins involves the Arg-Gly-Asp (RGD) tripeptide sequence. However, linear synthetic RGD peptides do not completely mimic the cell attachment activity of intact Fn or certain large Fn fragments. This suggests that the integrin-Fn interaction involves a more extended surface of Fn than that provided by the RGD sequence. To test this possibility, three novel monoclonal anti-Fn antibodies that inhibit its binding to a purified integrin, alpha IIb beta 3, were developed. The epitopes of these three antibodies mapped to a region at least 55 residues amino-terminal of the RGD sequence. Further, recombinant fragments of Fn containing these epitopes and lacking the RGD site also inhibited the binding of Fn to purified alpha IIb beta 3. These fragments, which spanned Fn residues 1359-1436, bound to alpha IIb beta 3 in a divalent cation-dependent manner. In addition, this region of Fn bound specifically to alpha IIb beta 3 on thrombin-stimulated but not resting platelets. These results demonstrate the presence of additional sequences in Fn that interact with integrin alpha IIb beta 3 and suggest that multiple sites in Fn are involved in its recognition by this integrin.     

Ligands "activate" integrin alpha IIb beta 3 (platelet GPIIb-IIIa)

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) binds fibrinogen via recognition sequences such as Arg-Gly-Asp (RGD). Fibrinogen binding requires agonist activation of platelets, whereas the binding of short synthetic RGD peptides does not. We now find that RGD peptide binding leads to changes in alpha IIb beta 3 that are associated with acquisition of high affinity fibrinogen-binding function (activation) and subsequent platelet aggregation. The structural specificities for peptide activation and for inhibition of ligand binding are similar, indicating that both are consequences of occupancy of the same site(s) on alpha IIb beta 3. Thus, the RGD sequence is a trigger of high affinity ligand binding to alpha IIb beta 3, and certain RGD-mimetics are partial agonists as well as competitive antagonists of integrin function.     

Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.     

Identification of a contact domain between echistatin and the integrin alpha(v)beta(3) by photoaffinity cross-linking.

The integrin alpha(v)beta(3) is the major receptor mediating the attachment of osteoclasts to the extracellular matrix in bone and plays a critical role in bone resorption and bone remodeling. Most of the ligands interacting with the alpha(v)beta(3) receptor contain an Arg-Gly-Asp (RGD) motif. Recently, we have identified two small RGD peptides, containing a benzophenone moiety at either the carboxyl or amino terminus, that photo-cross-linked within the beta(3)[99-118] [Bitan, G., et al. (1999) Biochemistry 38, 3414-3420] or the beta(3)[167-171] [Bitan, G., et al. (2000) Biochemistry 39, 11014-11023] sequence, respectively, of the alpha(v)beta(3) receptor in a selective fashion. Here, we report the synthesis of a photoreactive analogue of echistatin (a 49-amino acid peptide), a potent RGD-containing antagonist of the alpha(v)beta(3) receptor both in vitro and in vivo. This bioactive analogue is substituted at position 45 with a p-benzoyl moiety (pBz(2)), located within the flexible C-terminal domain and removed 20 amino acid residues from the R(24)GD(26) triad. This C-terminal domain was reported to contribute to receptor binding affinity by acting as an auxiliary binding site. The radiolabeled (125)I-[Arg(35),Lys(45)(N(epsilon)-pBz(2))]-echistatin photo-cross-links effectively to a site within the beta(3)[209-220] sequence. Residues in this domain have been reported to be part of the metal ion-dependent adhesion site (MIDAS). Receptor fragments overlapping this domain were reported to bind to fibrinogen and block fibrinogen binding to alpha(IIb)beta(3), the platelet integrin receptor. Taken together, position 45 in echistatin, located within an auxiliary binding site in echistatin, cross-links to a site distinct from the two previously reported sites, beta(3)[99-118] and beta(3)[167-171], which cross-link to photophores flanking the RGD triad. These cross-linking data support the hypothesis that the ligand-bound conformation of the integrin beta(3) subunit differs from the known conformation of I domains.     

Mapping of heparin/heparan sulfate binding sites on αvβ3 integrin by molecular docking

Heparin/heparan sulfate interact with growth factors, chemokines, extracellular proteins, and receptors. Integrins are αβ heterodimers that serve as receptors for extracellular proteins, regulate cell behavior, and participate in extracellular matrix assembly. Heparin binds to RGD‐dependent integrins (αIIbβ3, α5β1, αvβ3, and αvβ5) and to RGD‐independent integrins (α4β1, αXβ2, and αMβ2), but their binding sites have not been located on integrins. We report the mapping of heparin binding sites on the ectodomain of αvβ3 integrin by molecular modeling. The surface of the ectodomain was scanned with small rigid probes mimicking the sulfated domains of heparan sulfate. Docking results were clustered into binding spots. The best results were selected for further docking simulations with heparin hexasaccharide. Six potential binding spots containing lysine and/or arginine residues were identified on the ectodomain of αvβ3 integrin. Heparin would mostly bind to the top of the genu domain, the Calf‐I domain of the α subunit, and the top of the β subunit of RGD‐dependent integrins. Three spots were close enough from each other on the integrin surface to form an extended binding site that could interact with heparin/heparan sulfate chains. Because heparin does not bind to the same integrin site as protein ligands, no steric hindrance prevents the formation of ternary complexes comprising the integrin, its protein ligand, and heparin/heparan sulfate. The basic amino acid residues predicted to interact with heparin are conserved in the sequences of RGD‐dependent but not of RGD‐independent integrins suggesting that heparin/heparan sulfate could bind to different sites on these two integrin subfamilies. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural requirements of echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins

There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of alpha(IIb)beta(3), alpha(v)beta(3) and alpha(5)beta(1), and eristostatin, a similar disintegrin selectively inhibiting alpha(IIb)beta(3). In order to identify echistatin motifs required for selective recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins, we expressed recombinant echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The results showed that Asp(27) and Met(28) support recognition of both alpha(v)beta(3) and alpha(5)beta(1). Replacement of Met(28) with Asn completely abolished echistatin's ability to recognize each of the integrins, while replacement of Met(28) with Leu selectively decreased echistatin's ability to recognize alpha(5)beta(1) only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT exhibited new activity with alpha(5)beta(1), which was 10-20-fold higher than that of wild type eristostatin. A hypothesis is proposed that the C terminus of echistatin interacts with separate sites on beta(1) and beta(3) integrin molecules.     

Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells

Endothelial cells and activated platelets express integrin-type receptors responsible for adhesion to fibrinogen. We have located distinct integrin-directed endothelial cell and platelet attachment sites on immobilized fibrinogen using a combination of synthetic peptides, fibrinogen fragments, and specific anti-peptide monoclonal antibodies. Endothelial cells exclusively recognize an Arg-Gly-Asp-containing site near the C-terminus of the alpha chain (alpha residues 572-574) but fail to recognize the Arg-Gly-Asp sequence in the N-terminal region of the same chain (alpha residues 95-97). In contrast, platelets do not require either Arg-Gly-Asp sequence for binding to intact fibrinogen and are capable of recognizing, in addition to the alpha 572-574 sequence, a site at the C-terminus of the gamma chain (gamma residues 400-411). These data suggest a molecular mechanism whereby platelets and endothelial cells interact with distinct sites on the fibrinogen molecule during hemostasis and wound healing.     

Functional classification of ADAMs based on a conserved motif for binding to integrin alpha 9beta 1: implications for sperm-egg binding and other cell interactions

ADAMs (a disintegrin and metalloproteases) are members of the metzincin superfamily of metalloproteases. Among integrins binding to disintegrin domains of ADAMs are alpha(9)beta(1) and alpha(v)beta(3), and they bind in an RGD-independent and an RGD-dependent manner, respectively. Human ADAM15 is the only ADAM with the RGD motif in the disintegrin domain. Thus, both integrin alpha(9)beta(1) and alpha(v)beta(3) recognize the ADAM15 disintegrin domain. We determined how these integrins recognize the ADAM15 disintegrin domain by mutational analysis. We found that the Arg(481) and the Asp-Leu-Pro-Glu-Phe residues (residues 488-492) were critical for alpha(9)beta(1) binding, but the RGD motif (residues 484-486) was not. In contrast, the RGD motif was critical for alpha(v)beta(3) binding, but the other residues flanking the RGD motif were not. As the RX(6)DLPEF alpha(9)beta(1) recognition motif (residues 481-492) is conserved among ADAMs, except for ADAM10 and 17, we hypothesized that alpha(9)beta(1) may recognize disintegrin domains in all ADAMs except ADAM10 and 17. Indeed we found that alpha(9)beta(1) bound avidly to the disintegrin domains of ADAM1, 2, 3, and 9 but not to the disintegrin domains of ADAM10 and 17. As several ADAMs have been implicated in sperm-oocyte interaction, we tested whether the functional classification of ADAMs, based on specificity for integrin alpha(9)beta(1), applies to sperm-egg binding. We found that the ADAM2 and 15 disintegrin domains bound to oocytes, but the ADAM17 disintegrin domain did not. Furthermore, the ADAM2 and 15 disintegrin domains effectively blocked binding of sperm to oocytes, but the ADAM17 disintegrin domain did not. These results suggest that oocytes and alpha(9)beta(1) have similar binding specificities for ADAMs and that alpha(9)beta(1), or a receptor with similar specificity, may be involved in sperm-egg interaction during fertilization. As alpha(9)beta(1) is a receptor for many ADAM disintegrins and alpha(9)beta(1) and ADAMs are widely expressed, alpha(9)beta(1)-ADAM interaction may be of a broad biological importance.     

Function-blocking integrin alphavbeta6 monoclonal antibodies: distinct ligand-mimetic and nonligand-mimetic classes

We have generated a panel of potent, selective monoclonal antibodies that bind human and mouse alpha(v)beta(6) integrin with high affinity (up to 15 pm). A subset of these antibodies blocked the binding of alpha(v)beta(6) to the transforming growth factor-beta1 latency-associated peptide with IC(50) values as low as 18 pm, and prevented the subsequent alpha(v)beta(6)-mediated activation of transforming growth factor-beta1. The antibodies also inhibited alpha(v)beta(6) binding to fibronectin. The blocking antibodies form two biochemical classes. One class, exemplified by the ligand-mimetic antibody 6.8G6, bound to the integrin in a divalent cation-dependent manner, contained an RGD motif or a related sequence in CDR3 of the heavy chain, was blocked by RGD-containing peptides, and was internalized by alpha(v)beta(6)-expressing cells. Despite containing an RGD sequence, 6.8G6 was specific for alpha(v)beta(6) and showed no cross-reactivity with the RGD-binding integrins alpha(v)beta(3), alpha(v)beta(8),or alpha(IIb)beta(3). The nonligand-mimetic blocking antibodies, exemplified by 6.3G9, were cation-independent, were not blocked by RGD-containing peptides, were not internalized, and did not contain RGD or related sequences. These two classes of antibody were unable to bind simultaneously to alpha(v)beta(6), suggesting that they may bind overlapping epitopes. The "ligand-mimetic" antibodies are the first to be described for alpha(v)beta(6) and resemble those described for alpha(IIb)beta(3). We also report for the first time the relative abilities of divalent cations to promote alpha(v)beta(6) binding to latency-associated peptide and to the ligand-mimetic antibodies. These antibodies should provide valuable tools to study the ligand-receptor interactions of alpha(v)beta(6) as well as the role of alpha(v)beta(6) in vivo.     

Targeting integrins: insights into structure and activity of cyclic RGD pentapeptide mimics containing azabicycloalkane amino acids

A small library of cyclic RGD pentapeptide mimics incorporating stereoisomeric 5,6- and 5,7-fused bicyclic lactams was synthesized. This library was found to contain high-affinity ligands for the alpha(v)beta3 integrin. The aim of this study was to investigate activity, selectivity, and structure of these ligands in order to identify new specific alpha(v)-integrin antagonists that could be evaluated as tumor angiogenesis inhibitors. In vitro screening, including receptor-binding assays to purified alpha(v)beta3, alpha(v)beta5, and alpha5beta1 integrins, and platelet aggregation assay, revealed ST1646 as a potent, highly selective alpha(v)beta3/alpha(v)beta5 integrin antagonist. Structure determination of the cyclic RGD pentapeptide mimics performed by a combination of NMR spectroscopy, and molecular mechanics and dynamics calculations showed a strong dependence of the RGD cyclopeptide conformation on lactam ring size and stereochemistry. ST1646 revealed the highest ability within the library to adopt the proper RGD orientation required for binding to the alpha(v)beta3 integrin, as deduced from the recently solved crystal structure of the extracellular segment of integrin alpha(v)beta3 in complex with a cyclic pentapeptide ligand.     

A cluster of basic amino acid residues in the gamma370-381 sequence of fibrinogen comprises a binding site for platelet integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa)

Adhesive interactions of platelet integrin alpha(IIb)beta3 with fibrinogen and fibrin are central events in hemostasis and thrombosis. However, the mechanisms by which alpha(IIb)beta3 binds these ligands remain incompletely understood. We have recently demonstrated that alpha(IIb)beta3 binds the gamma365-383 sequence in the gammaC-domain of fibrin(ogen). This sequence contains neither the AGDV nor the RGD recognition motifs, known to bind alpha(IIb)beta3, suggesting the different specificity of the integrin. Here, using peptide arrays, mutant fibrinogens, and recombinant mutant gammaC-domains, we have examined the mechanism whereby alpha(IIb)beta3 binds gamma365-383. The alpha(IIb)beta3-binding activity was localized within gamma370-381, with two short sequences, gamma370ATWKTR375 and gamma376WYSMKK381, being able to independently bind the integrin. Furthermore, recognition of alpha(IIb)beta3 by gamma370-381 depended on four basic residues, Lys373, Arg375, Lys380, and Lys381. Simultaneous replacement of these amino acids and deletion of the gamma408AGDV411 sequence in the recombinant gammaC-domain resulted in the loss of alpha(IIb)beta3-mediated platelet adhesion. Confirming the critical roles of the identified residues, abnormal fibrinogen Kaiserslautern, in which gammaLys380 is replaced by Asn, demonstrated delayed clot retraction and impaired alpha(IIb)beta3 binding. Also, a mutant recombinant fibrinogen modeled after the naturally occurring variant Osaka V (gammaArg375 --> Gly) showed delayed clot retraction and reduced binding to purified alpha(IIb)beta3. These results identify the gamma370-381 sequence of fibrin(ogen) as the binding site for alpha(IIb)beta3 involved in platelet adhesion and clot retraction and define the new recognition specificity of this integrin.     

Structural basis for distinctive recognition of fibrinogen gammaC peptide by the platelet integrin alphaIIbbeta3

Hemostasis and thrombosis (blood clotting) involve fibrinogen binding to integrin alpha(IIb)beta(3) on platelets, resulting in platelet aggregation. alpha(v)beta(3) binds fibrinogen via an Arg-Asp-Gly (RGD) motif in fibrinogen's alpha subunit. alpha(IIb)beta(3) also binds to fibrinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the gamma subunit (gammaC peptide). These distinct modes of fibrinogen binding enable alpha(IIb)beta(3) and alpha(v)beta(3) to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin alpha(IIb)beta(3)-gammaC peptide interface, and, for comparison, integrin alpha(IIb)beta(3) bound to a lamprey gammaC primordial RGD motif. Compared with RGD, the GAKQAGDV motif in gammaC adopts a different backbone configuration and binds over a more extended region. The integrin metal ion-dependent adhesion site (MIDAS) Mg(2+) ion binds the gammaC Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca(2+) ion binds the gammaC C terminus, revealing a contribution for ADMIDAS in ligand binding. Structural data from this natively disordered gammaC peptide enhances our understanding of the involvement of gammaC peptide and integrin alpha(IIb)beta(3) in hemostasis and thrombosis.     

Platelet receptor recognition domains on the alpha chain of human fibrinogen: structure-function analysis

We have previously shown that the alpha chain of human fibrinogen interacts directly with ADP-activated human platelets [Hawiger, J., Timmons, S., Kloczewiak, M., Strong, D. D., & Doolittle, R. F. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2068]. Now, we report that platelet receptor recognition domains are localized on two CNBr fragments of the human fibrinogen alpha chain. They encompass residues 92-147 and 518-584, which inhibit 125I-fibrinogen binding to ADP-stimulated platelets. The inhibitory CNBr fragment alpha 92-147 contains the RGD sequence at residues 95-97. Synthetic peptides encompassing this sequence were inhibitory while peptide 99-113 lacking the RGD sequence was inactive. The synthetic peptide RGDF, corresponding to residues alpha 95-98, inhibited the binding of 125I-fibrinogen to ADP-treated platelets (IC50 = 2 microM). However, the peptides containing sequence RGDF, with residues preceding Arg95 or following Phe98, were less inhibitory. It appears that the sequence alpha 95-98 constitutes a platelet receptor recognition domain which is constrained by flanking residues. The second inhibitory CNBr fragment, alpha 518-584, also contains the sequence RGD at positions 572-574. Synthetic peptides overlapping this sequence were inhibitory, while peptides lacking the sequence RGDS were not reactive. Thus, another platelet reactive site on the alpha chain encompasses residues 572-575 containing sequence RGDS. In conclusion, the platelet receptor recognition domains on the human fibrinogen alpha chain in the amino-terminal and in the carboxy-terminal zones contain the ubiquitous cell recognition sequence RGD shared with other known adhesive proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

A spontaneous mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding site.

This work characterizes a mutant integrin alpha IIb beta 3 (glycoprotein (GP) IIb-IIIa) from a thrombasthenic patient, ET, whose platelets fail to aggregate in response to stimuli. The nature of defect was defined by the reduced ability of synthetic peptide ligands, corresponding to the carboxyl terminus of the fibrinogen gamma chain (gamma 402-411) and Arg-Gly-Asp (RGD), to increase the binding of the occupancy-dependent anti-LIBS1 antibody to mutant alpha IIb beta 3 and the reduced binding of mutant alpha IIb beta 3 to an immobilized RGD peptide. In addition, ET's platelets failed to bind the ligand-mimetic monoclonal anti-alpha IIb beta 3, PAC1. DNA sequence analysis of amplified ET genomic DNA revealed a single G----A base change which encoded substitution of R214 by Q in mature beta 3. Introduction of this point mutation into recombinant wild type alpha IIb beta 3 expressed in Chinese hamster ovary cells reproduced the ET platelet alpha IIb beta 3 deficits in binding of fibrinogen, mAb PAC1, and synthetic peptide ligands. Furthermore, substitution of R214 by Q in the synthetic peptide containing the sequence of beta 3(211-222) resulted in decreased ability of this peptide to block fibrinogen binding to purified alpha IIb beta 3. These findings suggest that substitution of beta 3 R214 by Q is responsible for the functional defect in alpha IIb beta 3 and that R214 is proximal to or part of a ligand binding domain in alpha IIb beta 3.