A Mutation in the  Subunit of the Platelet Integrin _IIbβ₃ Identifies a Novel Region Important for Ligand Binding

PLATELET ADHESIVE interactions mediated by the integrin α_IIbβ₃ are fundamental to the maintenance of normal hemostasis, as illustrated by the inherited bleeding disorder Glanzmann’s thrombasthenia. Platelets from patients with this disease either lack the α_IIbβ₃receptor, express reduced levels of the receptor, or express receptor variants that lack ligand binding function.1 In areas of vessel or tissue trauma, the α_IIbβ₃receptor initiates the formation of a platelet-based plug by binding to soluble fibrinogen, which subsequently forms a bridge to other platelets and facilitates platelet aggregation. Understanding the precise molecular mechanisms underlying the structural basis of ligand-receptor interaction is an important step towards the modulation of platelet function and can provide new insights into the development of novel antithrombotic therapies. Furthermore, understanding the molecular basis of this interaction by α_IIbβ₃, the prototypic integrin could provide insights into other integrin-ligand interactions in a diverse range of physiological processes.

Multiple ligand contact sites have been identified on α_IIbβ₃. Studies of natural receptor variants, cross-linking of ligand mimetic peptides, and site-directed mutagenesis studies have provided convincing evidence for the importance of two discontinuous regions in the β₃subunit, D119-S123 and D217-E220, in the ligand binding function of α_IIbβ₃.2-8 The importance of this region of β₃ appears to be due to its structural similarity to the integrin α subunit I-domain,9 which has been demonstrated to be critically involved in the ligand binding function of those integrins that contain this structure.10This region of β₃ may also function as a ligand/cation-binding MIDAS-like domain.8 The homologous region of other integrin β subunits is similarly critical for ligand binding function, substantiating that the amino terminal region of integrin β subunits is either directly involved in or structurally important for ligand binding receptor function.11-13 

Potential ligand contact sites on integrin α subunits have been identified by mapping epitopes of inhibitory monoclonal antibodies (MoAbs) and subsequent site-directed mutagenesis studies.14-16 Residues of α_IIb implicated in ligand binding function have also been identified by peptide studies17-19 and by the characterization of mutations present in patients with Glanzmann’s thrombasthenia. However, in contrast to many of the mutations identified in β₃, nearly all of the naturally occurring mutations identified in α_IIb reduce α_IIbβ₃ receptor expression to very low or undetectable levels.1 This suggests that the processing or structural stability of α_IIb is very sensitive to substitutions. The limited occurrence of well-expressed natural receptor variants containing α_IIb mutations has hindered the identification of specific residues in α_IIb that are critical to the ligand binding function of the receptor. Despite these difficulties, secondary structure predictions have been used to identify several residues in α_IIb whose substitutions blocked ligand binding but did not significantly affect expression of α_IIbβ₃.20 To overcome the inefficiencies of mutagenesis approaches in the absence of structural data and the rarity of Glanzmann’s thrombasthenia, we recently described a novel method for the identification of induced mutations that affect α_IIbβ₃ ligand binding function.21 The advantage of this method is its capacity to identify mutations that abolish ligand binding function but do not affect receptor expression. The present study identified a region in α_IIb that is important for ligand binding and is consistent with the predicted binding sites in the recently proposed structural model22 for the integrin α subunits.

The IgM_κ murine antibody PAC1 was kindly provided by Dr Sanford Shattil (The Scripps Research Institute, La Jolla, CA). PAC1 is an activation-specific, fibrinogen-mimetic MoAb that has been extensively characterized elsewhere.23 The α_IIbβ₃ complex-specific MoAbs AP224 and OPG225 were provided by Dr Thomas Kunicki (The Scripps Research Institute). The α_IIbβ₃ complex-specific MoAbs 4F10 and 2G12 were kindly provided by Dr Virgil Woods (University of California, San Diego, CA). The α_IIbβ₃ complex-specific MoAb D57, the anti-β₃ MoAb 15, and the activating anti-β₃ MoAbs, anti-LIBS1, anti-LIBS2, and anti-LIBS6, have been described elsewhere.26-28 The anti-α_v specific MoAb 14229 was purchased from Chemicon (Temecula, CA). The anti-α_IIb MoAb 98DF6 was obtained from Dr Jari Ylänne (University of Helsinki, Helsinki, Finland). The MoAb D57 was biotinylated using biotin-N-hydroxysuccinimide (Pierce, Rockford, IL) according to the manufacturer’s instructions. Fluorescein isothiocyanate-conjugated goat antimouse IgM and IgG were obtained from Biosource (Camarillo, CA). The α_IIbβ₃peptidomimetic Ro43-505430 was provided by Beat Steiner (Hoffmann-La Roche, Basel, Switzerland). The GRGDSPK-sepharose column was prepared as described.31 

The expression constructs pc3a and CD2b encoding wild-type β₃ and wild-type α_IIb, respectively, have been previously described.8,32 A 3.3-kb fragment of α_IIb containing the entire coding sequence and the 3′ untranslated region was excised from CD2b by digestion withXba I and ligated to the expression vector pcDNA3 (Invitrogen, San Diego, CA). The resulting construct was designated pc2b. The pCDM8 expression vector encoding wild-type α_vhas been previously described.4 An insert containing the entire coding sequence of α_v was removed from pCDM8 byXba I digestion and ligated to Xba I-digested pcDNA3. This construct was designated pcα_v. Construction of the expression plasmids encoding the chimeric subunits α_IIbα_6A and β₃β₁ has been described elsewhere.28 These chimeric plasmids contain the extracellular and transmembrane domains of human α_IIb or β₃ fused to the cytoplasmic domains of α_6Aand β₁, respectively. CD3hyg is a derivative of CD3a32 containing the hygromycin resistance gene. Amino acid residues of α_IIb are numbered, with the leucine residue at the amino terminus of the mature protein being residue number 1.

The chimeric α subunit, designated pcα_vα_IIb, consists of the backbone of α_v from which amino acids 181-223 were removed and replaced with the corresponding amino acids of exon 5 of α_IIb (amino acids G193-W235).33 It was constructed using the megaprimer method34 and three consecutive rounds of amplification. Chimeric oligonucleotide primers were constructed in which the 5′ ends contained α_vsequences (30 bp for each primer) and the 3′ ends contained the sequences either of the 5′ or the 3′ end of exon 5 of α_IIb (27 and 29 bp, 5′ and 3′ primer, respectively). The first round of polymerase chain reaction (PCR) used pc2b as the template and generated the 129-bp exon 5 of α_IIb containing 30 bp of α_v sequence on both the 5′ and 3′ ends. This fragment was gel-purified and used as a 5′ megaprimer together with a 3′ α_vprimer corresponding to the sequence 5′-GAATAGCCAAAGCTTGGTGGCATGC-3′ in a second round of PCR using pcα_v as the template to generate a 712-bp fragment containing exon 5 of α_IIb fused to 3′ α_v sequences. The 712-bp fragment was used as a 3′ megaprimer along with a 5′ α_v primer corresponding to the sequence 5′-CCGaGtaagCTTCGGCGATGGCTTTTCCGC-3′ in a final round of PCR with pcα_v as a template. The resulting 1,369-bp fragment contained exon 5 of α_IIb flanked by 5′ and 3′ α_v sequences. This PCR fragment was digested with HindIII and ligated to HindIII-digested pcα_v. The authenticity of the final construct was confirmed by DNA sequencing of the entire subcloned fragment.

Site-directed mutagenesis of selected α_IIb residues was performed using splice overlap extension as previously described.35 PCR-generated fragments containing α_IIb point mutations were digested with EcoRI andCla I, gel-purified, and ligated to EcoRI-ClaI–digested pc2b. All amplified fragments were sequenced in their entirety to verify the introduction of the mutation and the absence of any other substitutions.

The αβPy stable cell line was generated by transfecting CHO cells with three plasmids: α_IIbα_6A, β₃β₁, and a plasmid encoding the neomycin resistance gene.21 Chemical mutagenesis of the αβPy stable cell line was performed by treating 2 × 10⁶cells with 300 μg/mL ethyl methane sulfonate (EMS; Sigma, St Louis, MO) for 15 to 19 hours. After 1 week, approximately 1 × 10⁸ cells were harvested and ligand binding mutants were selected by flow cytometry.

Chemically mutagenized cells were individually sorted on a FAC-STAR Plus (Becton Dickinson, Mountain View, CA) using two-color flow cytometry with MoAbs D57 and PAC1 as described in detail.36 Cells that exhibited positive staining for receptor expression (D57 positive) but weak or absent PAC1 staining were individually sorted into 96-well plates and cultured for further analysis.

Surface expression of transfected integrins was analyzed by flow cytometry as described.27 PAC1 binding to transfected cells was analyzed by two-color flow cytometry as described.36PAC1 binding was analyzed only on the subset of cells positive for α_IIbβ₃ receptor expression that were gated using biotinylated D57. Some samples also contained 4 μmol/L of one of the anti-β₃ MoAbs anti-LIBS1, anti-LIBS2, or anti-LIBS6. These antibodies bind to distinct epitopes on β₃ and directly induce PAC1 binding to α_IIbβ₃.37 Other samples contained 2 μmol/L Ro43-5054.

Total cellular RNA was isolated from the selected cell lines using TRIzol (GIBCO/BRL, Grand Island, NY). cDNA was synthesized using oligo(dT) primers and the cDNA Cycle kit (Invitrogen). Resulting cDNA was amplified for 30 cycles with α_IIb-specific primers. The PCR products were directly sequenced using fluorescent automated sequencing (Applied Biosystems, Foster City, CA).

Cell lines were routinely passaged in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 1% nonessential amino acids, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. cDNAs were transfected into CHO cells with Lipofectamine (Life Technologies, Gaithersburg, MD) as previously described.36 cDNAs encoding α_vα_IIb, α_IIbD224V, or selected α_IIb mutants were transfected into CHO cells that had been previously transfected with the expression plasmid CD3hyg. Stably transfected cells were established by selection with 700 μg/mL G418. Clonal cell lines expressing α_vα_IIbβ₃ were selected by flow cytometry using the antihuman α_v-specific MoAb 142. Clonal lines expressing α_IIbD224Vβ₃ were sorted with the anti-α_IIbβ₃complex-specific antibody, D57.

Lysates of 3 × 10⁷ cells expressing either wild-type α_IIbβ₃, α_IIbD224Vβ₃, wild-type α_vβ₃, or α_vα_IIbβ₃ were prepared and applied to a GRGDSPK-Sepharose 4B column (1 mL bed volume) as described.31 The column was washed with 10 mL of lysis buffer and then eluted with 3 mL of buffer containing 1 mg/mL GRGDSPK. One-milliliter fractions were collected and resolved on 8% sodium dodecyl sulfate (SDS) polyacrylamide gels under nonreducing conditions. Proteins were transferred to nitrocellulose and blotted with the α_IIb-specific MoAb, 98DF6 (3.5 μg/mL), for the α_IIbβ₃ and α_IIbD224Vβ₃ receptors; with the α_v-specific MoAb, 142 (1:200 dilution of ascites), for the α_vβ₃ and α_vα_IIbβ₃ receptors; or with the β₃-specific MoAb, MoAb15 (3.5 μg/mL).

We have previously demonstrated that fusing the cytoplasmic domains of α_6A and β₁ to the extracellular and transmembrane domains of α_IIb and β₃, respectively, caused α_IIbβ₃ to assume the high-affinity state as defined by constitutive binding of the ligand mimetic antibody PAC1.28 Therefore, to isolate cell lines with disrupted integrin ligand binding function, CHO cells stably transfected with the active receptor variant α_IIbα_6Aβ₃β₁were exposed to the chemical mutagen, EMS. Mutagenized cells were then sorted by two-color flow cytometry to isolate those cells that failed to bind PAC1 but continued to express α_IIbβ₃ based on the binding of the MoAb D57 as described.21 Selected cells were individually sorted and subsequent analysis of the clonal cell lines grouped the mutants into three phenotypic classes: (1) ligand binding mutants, (2) integrin activation mutants, and (3) cellular activation mutants.21Individually sorted clonal lines that failed to bind PAC1 in the presence of the activating MoAb anti-LIBS6 were defined as ligand binding defective mutants. To identify which subunit contained a mutation resulting in the loss of ligand binding function, isolated cell lines with a ligand binding defect were transfected individually with the original α_IIbα_6A or β₃β₁ subunit and then reanalyzed for restoration of PAC1 binding. For most of the ligand binding defective mutants, PAC1 binding was restored after retransfection with the parental β subunit β₃β₁, indicating the causative mutation was present in the β subunit. In contrast, PAC1 binding was reconstituted in one cell line only after retransfection with α_IIbα_6A, indicating that the defect was likely contained in the α_IIb subunit. This cell line was subjected to further analysis.

Total RNA was isolated from this cell line and subjected to RT-PCR. The entire α_IIbα_6A subunit was amplified in three separate fragments and the resulting PCR products were sequenced directly. Analysis of the resulting sequence identified a single A→T mutation resulting in substitution of D224 by valine.

To confirm that this single amino acid substitution was responsible for the observed loss of ligand binding function, this mutation was introduced into wild-type α_IIb cDNA and transfected into cells stably expressing the wild-type β₃ subunit. Whether transiently or stably transfected, the α_IIbD224Vβ₃ receptor was expressed on the cell surface and bound a panel of complex-specific anti-α_IIbβ₃ MoAbs, including D57 (Fig 1A), 2G12, 4F10, and AP2, suggesting the mutation exerted minimal structural effects. However, cells expressing α_IIbD224Vβ₃ did not bind MoAb PAC1 in the presence or absence of several activating antibodies, including anti-LIBS6 (Fig 1D), anti-LIBS1, anti-LIBS2, and AP5. The inability of these MoAbs to activate PAC1 binding was not due to a loss of antibody epitopes, because all of these MoAbs bound to α_IIbD224Vβ₃, as assayed by flow cytometry (data not shown). Furthermore, cells expressing α_IIbD224Vβ₃ exhibited minimal binding of the activation-independent ligand mimetic MoAb OPG2 compared with cells expressing wild-type α_IIbβ₃ (Fig 1B). These results confirm that substitution of α_IIbD224 alone was sufficient for the functional defect.

The failure of α_IIbD224Vβ₃ to bind the α_IIbβ₃-specific ligands PAC1 and OPG2 could represent the loss of α_IIbβ₃-specific ligand recognition or the loss of ligand binding function in general. To discriminate between these two possibilities, the RGD recognition function of α_IIbD224Vβ₃ was examined by affinity chromatography. Lysates of α_IIbD224Vβ₃-expressing or wild-type α_IIbβ₃-expressing cells were applied to a GRGDSPK Sepharose column, washed, and then eluted with GRGDSPK peptide. Eluted fractions were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then immunoblotted with anti-α_IIb or anti-β₃ MoAbs (Fig 2). Wild-type α_IIbβ₃ bound to the matrix and was specifically eluted by GRGDSPK peptide. In contrast, the mutant α_IIbD224Vβ₃ showed only minimal binding to the affinity matrix. The failure of α_IIbD224Vβ₃ to bind to the RGD peptide indicates that this substitution results not only in the loss of binding of α_IIbβ₃-specific ligands such as PAC1 and OPG2, but also the loss of binding to ligands shared in common with other integrins.

To determine whether the observed functional defect was due to the substitution with valine, site-directed mutagenesis was used to replace D224 by alanine. When transiently transfected into CD3a cells, α_IIbD224A formed a complex with β₃ and was expressed on the cell surface as assayed by flow cytometry. However, similar to cells expressing the α_IIbD224Vβ₃mutant, cells expressing α_IIbD224Aβ₃ did not bind PAC1 in the presence or absence of the activating antibody, anti-LIBS6 (Fig 3A).

The region surrounding D224 consists of a number of amino acids containing oxygenated side chains, particularly serine. This grouping of serine residues resembles the alignment of Asp and Ser residues that constitute a portion of the MIDAS motif found in the I domains of certain integrin α subunits and in the related structure present in all integrin β subunits. A number of these serine residues (S220, S222, and S226) were replaced by alanine and the effect on ligand binding was examined after transient and stable transfection of CD3a cells. The receptors α_IIbS220Aβ₃, α_IIbS222Aβ₃, and α_IIbS226Aβ₃ were all expressed on the cell surface and retained their ability to bind MoAb PAC1 (Fig 3B through D). Interestingly, Ro43-5054 did not inhibit the binding of PAC-1 to α_IIbS222Aβ₃. The mechanism for this loss of inhibition is unknown.

Alignment of the integrin α subunits shows that the region corresponding to α_IIbD224 is not well conserved (Fig 4). Because other known ligand binding regions of the integrin receptors often exhibit a high degree of similarity, this lack of conservation suggested that this region might be important in determining the ligand recognition specificity of α_IIbβ₃. To test this hypothesis, a chimeric α subunit was constructed by inserting exon 5 of α_IIb, which contains D224, into the corresponding location in the related α_v subunit. The α_vα_IIb chimeric subunit was stably expressed in CD3a cells and assayed for the capacity to bind the α_IIbβ₃-specific ligand PAC1. The chimeric receptor was well expressed on the cell surface (Fig 5A); however, these cells did not exhibit binding to PAC1 in the presence or absence of the activating antibody anti-LIBS6 (Fig 5B). Similarly, binding to the activation-independent ligand mimetic antibody OPG2 was not observed (data not shown). Introduction of the α_IIb sequence into α_v did not disrupt ligand binding in general, because the chimeric receptor retained the capacity to bind to a RGD affinity matrix (Fig 5C).

We report the identification of a novel mutation in the integrin α_IIb subunit (D224→V) that results in loss of α_IIbβ₃ adhesion receptor function. Unlike most of the mutations identified in the α_IIb subunit, this mutation did not significantly affect expression of α_IIbβ₃ on the cell surface. However, α_IIbD224Vβ₃ did not bind the activation-dependent ligand mimetic antibody, PAC1, and bound only minimally to the activation-independent ligand mimetic antibody, OPG2. Substitution of D224 by alanine also resulted in a loss of receptor function. Insertion of exon 5 of α_IIb containing D224 into the backbone of the α_v subunit did not enable the resulting chimera to bind α_IIbβ₃-specific ligands; however, it did not interfere with ligand binding to α_vβ₃. These data suggest that α_IIbD224 may play an important role in ligand binding specific to α_IIbβ₃.

An unbiased genetic approach was used to identify chemically induced mutations that result in the loss of α_IIbβ₃ligand binding activity. A key feature of this approach is the ability to isolate cells that expressed α_IIbβ₃ on the cell surface but failed to bind the activation-dependent, ligand mimetic antibody PAC1.21 Whereas the majority of the isolated clones contained substitutions in the β₃subunit, PAC1 binding was restored in one clone only after retransfection with the parental α subunit. This mutant receptor contained a single basepair change resulting in substitution of D224 by valine. This receptor was expressed on the cell surface as assayed with a panel of anti α_IIbβ₃ MoAbs, indicating that the substitution likely had minimal effects on the processing or structure of this subunit. This finding is in sharp contrast to most of the identified naturally occurring mutations in α_IIb that significantly reduce or abolish receptor expression.1The effect of the mutation at D224 on ligand binding to α_IIbβ₃ was not restricted to α_IIbβ₃-specific ligands such as PAC1 and OPG2. This mutation also significantly decreased the binding of a RGD peptide. Thus, this mutation affects the binding of ligands specific to α_IIbβ₃ and the binding of ligands shared in common with other integrins.

D224 is located in a group of serine residues distributed in a pattern that resembles a portion of the MIDAS motif known to be important for cation and ligand binding in the I domain of integrin α subunits and in a related motif in the integrin β subunits.8,9,11-13,38 39 However, substitution of several of these serine residues (S220, S222, and S226) with alanine did not result in the loss of PAC1 binding, suggesting that they do not directly play a critical role in the ligand binding process. Surprisingly, the substitution of S222 did appear to inhibit the capacity of the peptidomimetic Ro43-5054 to block PAC1 binding. The mechanism of the effect of substitution of D224 on ligand binding is unclear. It is unlikely to be the result of a gross structural alteration, because the mutant receptor bound a panel of complex-specific anti-α_IIbβ₃ antibodies. Other possibilities include direct interaction of D224 with ligand or, alternatively, through an indirect method by interacting cooperatively with other regions present in α_IIb or in the β₃ subunit. The precise functional assignment of this residue cannot be defined without high resolution structural data for the α_IIbβ₃ receptor.

The ligand recognition specificity of the integrins is determined in large part by the α subunits. In the case of α_IIbβ₃, the specificity of ligand recognition maps to the first 334 residues of the α_IIbsubunit.40 The location of D224 is consistent with this data. Alignment between the integrin α subunits demonstrates that the region surrounding D224 is not well conserved, suggesting that this region of α_IIb may be involved in a specific rather than general role of ligand recognition. Previous work from this laboratory had demonstrated that a larger region of α_IIb (ie, amino acids R140-P334) did not confer α_IIbβ₃ligand binding specificity to an α_vα_IIbchimeric α subunit.40 In the present study, a smaller chimera (α_IIb amino acids 193-235) was constructed consistent with α_IIb intron/exon boundaries. Although this chimeric integrin consisting of the α_v subunit and exon 5 of α_IIb was well expressed, it did not bind the α_IIbβ₃-specific ligand PAC1. Moreover, disruption of the native α_v region with the α_IIb sequences did not abolish the binding of RGD peptides to this chimera. Thus, the region of α_IIb that contains D224 may fully substitute for the analogous α_vsequences or the analogous α_v region is not similarly important for ligand binding to α_vβ₃. Although the lack of conservation between the α subunits in this region makes it difficult to accurately align residues with D224, these data do indicate that D224 is important for ligand binding function of α_IIbβ₃.

A structural model for the N-terminal approximately 440 amino acids of the integrin α subunits has recently been proposed.22This model predicts that the seven homologous sequence repeats found in the α subunits adopt the fold of a β-propeller domain. Enzymes with known β-propeller folds have their active sites at the top of the β-propeller, typically where adjacent loops run in opposite directions, such as seen between strands 2 and 3 within a β sheet and strands 4 and 1 between two sheets. The residue identified in this study, α_IIbD224, lies between repeats III and IV in the α_IIb subunit and is located in the large 4-1 loop region located at the top of the β propeller between the W3 and W4 β sheets. In a previous study, the α_IIb region, G184-193, was implicated as important for ligand binding and is located in the adjacent 2-3 loop found in the W3 sheet at the top of the β-propeller.20 Thus, these two loop regions could act synergistically to form a portion of the ligand binding pocket between the α_IIb and β₃ subunits. The recent identification of an α_IIb L183→P mutation in a Glanzmann’s thrombasthenia patient41 that produces quantitative and qualitative abnormalities in the receptor also substantiates the importance of this region in ligand binding. The identification of residues critical for α₄β₁-ligand interactions14 and for the binding of fibronectin to the integrin α₅β₁16 further supports a role for the loop structures of the α subunits on the upper face of the predicted β-propeller model in ligand binding specificity.

The region identified in this study is located between the homologous repeats III and IV found in all integrin α subunits. Interestingly, this region is known to be alternatively spliced in α₆and α₇ and has been postulated to be alternatively spliced in α₃.42,43 In α₆ and α₇, alternative splicing produces two variants, one of which (the α₇X2 variant) contains a Asp residue homologous to D224 in α_IIb (see Fig 4). The α₇ splice variants are both developmentally regulated and tissue specific. Although no functional significance has yet been determined for these different variants, it has been speculated that this region may be involved in ligand specificity and/or different ligand binding affinities. Alternative splicing between the third and fourth repeats has also been reported for the Drosophila integrin PS2α subunit.44 45 Together, the differences among such variants could suggest a general function for this region in ligand recognition specificity consistent with the low conservation of this region among different α subunits.

In summary, this study has identified a novel region in α_IIb that is important for ligand binding. This location of this region is within a predicted ligand binding site in the β propeller model proposed for the α subunits. This region is not highly conserved among the α subunits, and substitutions in the analogous regions of α_v did not affect ligand binding function of α_vβ₃. These results indicate that this region of α_IIb may play a role in ligand binding that is specific to α_IIbβ₃.