The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are dependent on the interaction of the leukocyte integrin Mac-1 (αMβ2, CD11b/CD18) and the platelet counter receptor glycoprotein (GP) Ibα. Previous studies have established a central role for the I domain, a stretch of ∼200 amino acids within the αM subunit, in the binding of GP Ibα. This study was undertaken to establish the molecular basis of GP Ibα recognition by αMβ2. The P201–K217 sequence, which spans an exposed loop and amphipathic α4 helix in the three-dimensional structure of the αMI domain, was identified as the binding site for GP Ibα. Mutant cell lines in which the αMI domain segments P201–G207 and R208–K217 were switched to the homologous, but non-GP Ibα binding, αL domain segments failed to support adhesion to GP Ibα. Mutation of amino acid residues within P201–K217, H210–A212, T213–I215, and R216–K217 resulted in the loss of the binding function of the recombinant αMI domains to GP Ibα. Synthetic peptides duplicating the P201–K217, but not scrambled versions, directly bound GP Ibα and inhibited αMβ2-dependent adhesion to GP Ibα and adherent platelets. Finally, grafting critical amino acids within the P201–K217 sequence onto αL, converted αLβ2 into a GP Ibα binding integrin. Thus, the P201–K217 sequence within the αMI domain is necessary and sufficient for GP Ibα binding. These observations provide a molecular target for disrupting leukocyte–platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.
Adhesive interactions between vascular cells play important roles in orchestrating the inflammatory response. Recruitment of circulating leukocytes to vascular endothelium requires multistep adhesive and signaling events, including selectin-mediated attachment and rolling, leukocyte activation, and integrin-mediated firm adhesion and diapedesis that result in the infiltration of inflammatory cells into the blood vessel wall (1). Firm attachment is mediated by members of the β2 integrin family, LFA-1 (αLβ 2, CD11a/CD18), Mac-1 (αMβ2, CD11b/CD18), and p150,95 (αMβ2, CD11c/CD18), which bind to endothelial counter ligands (e.g., intercellular adhesion molecule [ICAM]-1; 2), endothelial-associated extracellular matrix proteins (e.g., fibrinogen; 3), or glycosaminoglycans (4).
Leukocyte recruitment and infiltration also occur at sites of vascular injury where the lining endothelial cells have been denuded and platelets and fibrin have been deposited. A similar sequential adhesion model of leukocyte attachment to and transmigration across surface-adherent platelets has been proposed (5). The initial tethering and rolling of leukocytes on platelet P-selectin (6) are followed by their firm adhesion and transplatelet migration, processes that are dependent on αMβ2 (5).
Our laboratory has focused on identifying the platelet counter receptor for αMβ2. Evaluation of the structural features of integrins provides insight into candidate platelet counter receptors for αMβ2. Integrins are heterodimeric proteins composed of one α and one β subunit. A subset of integrin α subunits, including αM, contains an inserted domain (I domain) of ∼200 amino acids that is implicated in ligand binding (7–9) and strikingly similar to the A domains of von Willebrand factor (vWf; 10), one of which, A1, mediates the interaction of vWf with its platelet receptor, the glycoprotein (GP) Ib-IX-V complex. Because of the similarity of the vWf A1 domain and the αMI domain, we hypothesized that GP Ibα might also be able to bind αMβ2 and reported that GP Ibα is indeed a constitutively expressed counter receptor for αMβ2 (11). Furthermore, under the conditions used in these studies, the predominant interaction between neutrophils and platelets appeared to be between αMβ2 and GP Ibα (11).
The αMI domain contributes broadly to the recognition of ligands by αMβ2 (12) and specifically to the binding of GP Ibα (11). This region has been implicated in the binding of ICAM-1 (13), iC3b (14), fibrinogen (12, 13), and neutrophil inhibitory factor (NIF; 15), as well as GP Ibα. Previous studies suggested that overlapping, but not identical, sites are involved in the recognition of iC3b, fibrinogen, and NIF (16, 17). Although the binding sites for iC3b, NIF, and fibrinogen in the αMI domain have been mapped extensively (18–23), the recognition site for GP Ibα is unknown.
In this study, we have localized the binding site for GP Ibα within the αMI domain. The strategy developed was based on the differences in the binding of GP Ibα to the αMI and αLI domains and involved several independent approaches, including screening of mutant cells, synthetic peptides, site-directed mutagenesis, and gain in function analyses. The binding site for GP Ibα was localized within the segment αM(P201–K217). The grafting of two amino acids within this segment into the αLI domain converted it to a GP Ibα–binding protein. Thus, a small segment that has a defined structure within the αMI domain is necessary and sufficient for GP Ibα binding.
Materials And Methods
The soluble extracellular region of GP Ibα (sGP Ibα; i.e., glycocalicin) was purified as we previously reported (11). Human fibrinogen depleted of plasminogen, vWf, and fibronectin was purchased from Enzyme Research Laboratories.
The CD11/CD18 mAbs used included the following: LPM19c, directed to the αMI domain (provided by K. Pulford, Radcliffe, Oxford, United Kingdom; 12); OKM1, directed to the αM subunit of human Mac-1 (American Type Culture Collection [ATCC]); M1/70, directed to the αM subunit mouse αMβ2 (ATCC; 24); CBRM1/5, an activation-specific αM reporter antibody (provided by T. Springer, Harvard Medical School, Boston, MA; 25); TS1/22, directed to the αL subunit of αLβ2 and capable of blocking ICAM-1 binding (ATCC); MEM-83, an activation-specific αL reporter antibody (Caltag); and IB4, a blocking mAb directed to the β2 subunit (ATCC). The stimulating CD18 mAb KIM127 (26) was provided by M. Robinson (Celltech Ltd., Slough, United Kingdom). mAb24, a β2 activation reporter antibody (27), was provided by N. Hogg (Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom).
Peptides were obtained from the W.M. Keck Biotechnology Resource Center at Yale University. The peptides were diluted in DMSO and stored at −80°C.
Cell Lines and Culture Conditions.
Segment Switches by Site-directed Mutagenesis.
To systematically define the GP Ibα–binding site in αM, a homologue-scanning mutagenesis strategy was implemented (20). Accordingly, guided by the crystal structure (8, 9), the hydrated surface of the αMI domain was replaced with sequences of the αLI domain in segments of 7–11 amino acids. To apply this approach to the αMI domain (∼200 amino acids), 16 segments were switched (20, 23). Site-directed mutagenesis of the αMI domain was performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene). The mutations introduced and the mutagenic primers used have been reported (23). The appropriate DNA sequence of the entire I domain (from I139 to A332) was confirmed for each mutant before transferring back into the αM subunit cDNA.
The expression vector pcDNA3.1 (Invitrogen) was used for cloning αM, αL, and β2 from human leukocyte cDNA library. 293 cells were transfected using the Lipofectamine 2000 reagent (Invitrogen) with 24 μg DNA/vessel for 4 h, according to the manufacturer's instructions. After transfection, the medium was replaced with full growth medium. The functional assays were prepared 48 h after transfection.
FACS® analyses were performed to assess the expression of wild-type and mutated forms of αMβ2 and αLβ2 on the surface of transfected 293 cells, as previously described (11). Platelet P-selectin expression was assessed using FITC-conjugated AK-4 or isotype control (BD Biosciences).
Preparation of Neutrophils and Platelets.
Neutrophils from wild-type (Mac-1+/+) and Mac-1–deficient (Mac-1−/−; 30) C57Bl/J6 mice were harvested and purified from the peritoneal cavity after the intraperitoneal injection of 1 ml sterile 3% thioglycollate broth, as previously described (11).
Venous blood was obtained from volunteers who had not consumed aspirin or other nonsteroidal antiinflammatory drugs for at least 10 d and was anticoagulated with 13 mM trisodium citrate, which also contained 100 nM prostaglandin E1. Platelet-rich plasma was prepared by centrifugation at 150 g for 10 min. Gel-filtered platelets were obtained by passage of platelet-rich plasma over a Sepharose-2B column in calcium-free Tyrode's-Hepes buffer containing 100 nM prostaglandin E1, as previously described (11).
Adherent cells were assayed by loading 293 cells and thioglycollate-elicited murine neutrophils with 1 μM 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF AM), according to the manufacturer's instructions (Molecular Probes). 105 cells/well were placed in 96-well microtiter plates coated with 10 μg/ml sGP Ibα or 10 μg/ml fibrinogen and blocked with 0.2% gelatin. Adhesion was stimulated with 20 ng/ml PMA or 5 μg/ml of the β2-stimulating mAb KIM 127. Plates were washed and adhesion was quantified by measuring the fluorescence of BCECF AM–loaded cells using a Cytofluor II fluorescence microplate reader (PerSeptive Biosystems). The effect of anti-αM mAb on adhesion was assessed by preincubating cells with 10 μg/ml LPM19c. The effect of peptides M1–M8 on adhesion was investigated by incubating the indicated peptide with sGP Ibα–coated wells for 30 min at 37°C before the addition of cells. Data are expressed as percent adhesion of control treatment.
Neutrophil Adhesion to Surface-adherent Platelets.
Neutrophil adhesion to surface-adherent platelets was investigated as previously described (11). The effect of αM peptides on leukocyte adhesion to platelets was examined by preincubating surface-adherent platelets with peptide (1–1,000 nM) or vehicle for 30 min at 37°C. Data are expressed as percent adhesion of control treatment.
Site-directed Mutagenesis, Expression, and Purification of I Domain Fusion Proteins.
The cDNAs of αMI domain (675 nucleotides, R115–S340), αLI domain (630 nucleotides, P120–S330), and mouse αMI domain (675 nucleotides, L115–S340) were cloned and inserted into the pGEX-5X-3 expression vector (22). All wild-type and mutant I domains were expressed as glutathione S-transferase (GST) fusion proteins. Mutations were created in these I domains and intact αL by oligonucleotide-directed mutagenesis. The selective introduction of the desired mutations into the I domains was confirmed by DNA sequence analyses. The GST-I domain fusion proteins were purified by adsorption onto glutathione-Sepharose 4B (Amersham Biosciences) and eluted with buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM glutathione, 2.5 mM CaCl2. Such preparations of the fusion proteins were ∼90% pure as assessed by SDS-PAGE.
GP Ibα Binding to the I Domains.
To test the interaction of the wild-type αM, αL, and chimeric I domains with biotinylated GP Ibα, 96-well plates (Immulon 4BX; Dynex Technologies Inc.) were coated with the I domains at 50 μg/ml and blocked with 2% BSA. sGP Ibα in 20 mM Tris-HCl, pH 7.6, containing 100 mM NaCl and 2 mM CaCl2, was added to the wells and incubated for 1 h at 37°C. After washing, bound GP Ibα was detected using avidin-alkaline phosphatase and p-nitrophenyl phosphate. Background reaction on BSA-coated wells was subtracted.
BIAcore Surface Plasmon Resonance Analysis.
Real-time protein–protein interactions were examined using surface plasmon resonance on a BIAcore 1000 (BIAcore AB). Immobilization of peptides was performed via thiol coupling onto a sensor chip C1 using 20 mM Tris, 150 mM NaCl, 2 mM MgCl2, pH 7.4, as running buffer at a flow rate of 5 μl min−1. Modified versions of peptides containing an amino-terminal cysteine residue were synthesized and then diluted in10 nM acetate buffer, pH 5.0 (coupling buffer). The peptide was loaded on the chip for 16 min and immobilization occurred via thiol disulfide exchange. Identical treatment was applied to the reference flow cell without peptide (control surface). For analysis, sGP Ibα diluted into running buffer was injected at a flow rate of 10 μl/min. Binding (resonance unit [RU]) was measured as a function of time(s). Binding data (after subtracting nonspecific background binding to the control surface) are presented as sensorgrams, binding curves, and Scatchard plots (31).
In Vitro Analysis of Cellular Adhesion under Laminar Flow Conditions.
The laminar flow chamber used in this assay has been described (32). 25 mm2 diameter glass coverslips (Assistent; Carolina Biological Supply Company) were coated with a solution of 20 μg/ml soluble P-selectin (provided by R. Camphausen, Genetics Institute, Cambridge, MA) and 40 μg/ml sGP Ibα for 2 h at room temperature. Nonspecific interactions were blocked with PBS containing 1% human serum albumin for 1 h at room temperature and with 0.1% Tween 20 immediately before use. The effect of M2 and scM2 peptides on THP-1 cell rolling and firm arrest was examined by incubating 50 μM of the peptide with the ligand-coated glass coverslip for 1 h at room temperature. To block PSGL-1 and αMβ2 interactions, THP-1 cells were incubated with 20 μg/ml KPL-1 (BD Biosciences) or 20 μg/ml LPM19c mAb, respectively, for 20 min before perfusion. αMβ2 was activated by treating cells with 10 μg/ml KIM127 for 5 min before perfusion. The rolling and firm adhesive events scored were ligand specific as confirmed in parallel determinations on control substrates coated with human serum albumin. Coverslips were inserted in the flow chamber and 0.6 × 106/ml THP-1 cells were drawn across at an estimated shear stress of 0.75 dynes/cm2 using a syringe pump (Harvard Apparatus). After 3 min of perfusion, the number of rolling and arrested cells was quantified in each of five random 10x fields (area ∼0.3 mm2) by an investigator blinded to treatment.
Data are presented as the mean ± SD or SEM. Groups were compared using the nonpaired t test. Rolling and arrest data were analyzed using repeated measure ANOVA with a Bonferroni corrective for multiple comparisons. P values <0.05 were considered significant.
Binding of GP Ibα to Mutant Cell Lines.
Our previous report indicated that the αMI domain serves as a recognition site for GP Ibα (11). The adhesion of αMβ2-bearing cells to sGP Ibα was inhibited by LPM19c, an mAb that binds to the αMI domain. To establish definitively that the αMI domain serves as a recognition site for GP Ibα, we transfected 293 cells with wild-type αMβ2, αLβ2, or a chimeric αLβ2 receptor that contained the I domain of αMβ2 (i.e., αL(IαM)β2-transfected 293 cells). αLβ2-transfected 293 cells did not adhere to sGP Ibα. In contrast, αL(IαM)β2-transfected 293 cells adhered robustly to sGP Ibα in a manner similar to αMβ2-transfected 293 cells, indicating that the αMI domain is required for adhesion to sGP Ibα. These data suggest that although αMβ2 and αLβ2 are highly homologous integrins, the lack of binding of sGP Ibα to αLβ2 might be the result of sequence and/or structural differences.
As the first step to define the binding site for GP Ibα within the αMI domain, mutant cell lines, each expressing a mutant αMβ2 in which a short αMI domain sequence, corresponding to a structural unit in the crystal structure (8, 9), was replaced for the corresponding region of the αLI domain, were tested for their adhesion to immobilized sGP Ibα. 16 segments were switched from their original αMI domain to their counterparts in αL (16, 20, 23). These segment swaps are placed throughout the P147–Q314 region of the αMI domain, D132–A318, and are intended to cover its entire hydrated surface (8, 9). For such experiments to be readily interpretable, cell lines expressing high and similar levels of wild-type and mutant receptors were selected by cell sorting using mAbs OKM1 and IB4 followed by cloning by limiting dilution. The expression of the receptors as assessed by the mean fluorescence intensity (MFI) in FACS® analyses differed by less than twofold from the internal wild-type control (not depicted).
The results of adhesion of 16 mutants to sGP Ibα are summarized in Fig. 1. The data are expressed as the percent adhesion of the wild-type αMβ2-expressing cells to sGP Ibα. Substitutions for the following regions of αMI domain abrogated adhesion: αM(P147–R152), αM(M153–T159), αM(E162–L170), αM(P201–G207), αM(R208–K217), αM(K245FG247), αM (ΔD248–Y252), αM(E253–R261), αM(D273–K279), αM(R281–I287), and αM(F297–T307). These cell lines form a group of negative mutants. As the essential control, the αLβ2-expressing cells adhered poorly to sGP Ibα, consistent with lack of interaction of αLβ2 with sGP Ibα.
The lack of adhesion of these mutants was not the result of decreased surface expression of the receptor because there was no correlation between the level of adhesion and expression. Specifically, surface expression of the αM(P201–G207) mutant was 1.5-fold higher than that of the cells expressing the wild-type αMβ2 cells, but adhesion was abrogated completely. Surface expression of negative mutants, αM(P147–R152), αM(R208–K217), αM(E253–R261), and αM(D273–K279) was very similar to that of the wild-type receptor. Adhesion of αM(E178–T185) and ΔE262G263 was partially affected. The maximal level of adhesion to sGP Ibα reached 69 and 64%, respectively, of wild-type αMβ2 cells. These two receptors were classified as intermediate mutants.
The following mutants supported the same or higher levels of adhesion to sGP Ibα compared with the cells expressing the wild-type αMβ2 receptor: αM(Q190–S197), αM(K231NAF234), αM(E262–G263), and αM(Q309–E314). The cell lines exhibiting adhesion similar to or greater than the wild-type αMβ2 cells were identified as positive mutants. Two of these positive mutants, αM(Q190–S197) and αM(K231NAF234), bound sGP Ibα better than wild-type αMβ2. Previous analyses using these mutants showed that they bound neither an αMβ2 activation-independent ligand, NIF (20), nor αMβ2 activation-dependent ligands, C3bi (17) and fibrinogen (23), to a greater extent than wild-type receptor. Thus, the enhanced binding of these mutants appears to be selective for sGP Ibα recognition. In further studies, we found that binding of the β2 activation–specific reporter mAb 24 was similar for wild-type and the αM (Q190–S197) and αM(K231NAF234) mutants. In contrast, increased binding of the αM activation–specific reporter CBRM1/5 to αM(K231NAF234) compared with wild-type αMβ2 and αM(Q190–S197) suggests possible conformation change of the αM(K231NAF234) mutant (not depicted). Together, these observations suggest that the activation states of αMβ2 may reflect the reporter ligand or mAb used, and these two mutants might be functionally activated with respect to sGP Ibα recognition.
Interaction of GP Ibα with the αMI Domain Peptides.
In subsequent analyses, we focused on the 11 negative mutants. A series of peptides (M1–M8) corresponding to the wild-type αMI domain sequences were synthesized and tested for their ability to interact with sGP Ibα. Because the sequences within several of the negative mutants were contiguous, the following linear peptides spanning these sequences were synthesized: P147–T159 (designated M1), P201–K217 (M2), K245–R261 (M3), D273–I287 (M4), F297–T307 (M5), Q190–S197 (M6), E162–L170 (M7), and E178–T185 (M8). All peptides correspond to negative mutant sequences except M6 and M8, which correspond to intermediate and positive mutant sequences, respectively.
Next, we examined the effect of these αMI domain peptides on αMβ2-dependent adhesion to sGP Ibα or fibrinogen (Table I). 50 μM peptides were preincubated in ligand-coated wells, αMβ2-expressing 293 cells were added, and adhesion was stimulated with KIM 127. Mac-1 293 cells adhered to sGP Ibα and fibrinogen, and this adhesion was blocked by the anti-CD11b mAb LPM19c, indicating that adhesion is predominantly αMβ2 dependent (Table I). Peptide M2 inhibited adhesion to sGP Ibα (percent inhibition = 74 ± 13; P < 0.01). Interestingly, M2 had no effect on cellular adhesion to fibrinogen. All other peptides had minimal or no effect on αMβ2-expressing 293 cell adhesion to sGP Ibα or fibrinogen. M2, but not a scrambled version (scM2), blocked αMβ2-dependent adhesion to sGP Ibα in a dose-dependent manner (IC50 = 20 μM; Fig. 2). Taken together, these observations suggest that the αMI domain sequence corresponding to M2 (P201–K217), which spans an exposed loop and amphipathic α4 helix in the three-dimensional structure of the αMI domain, contributes to αMβ2 binding of GP Ibα.
Real-Time Detection of Protein–Protein Interaction by BIAcore Analyses Reveals Specific, Direct Binding of GP Ibα to M2 Peptide.
To examine direct interactions between GP Ibα and M2, we used an in vitro real-time binding assay using surface plasmon resonance (BIAcore). M2 and scM2 peptides were modified by addition of an amino-terminal cysteine and immobilized via thiol coupling on a C1 sensor chip. Cys-M2, but not Cys-scM2, inhibited αMβ2-dependent adhesion to sGP Ibα, verifying that cysteine addition did not adversely affect peptide structure (Table I). Increasing concentrations of purified sGP Ibα in running buffer containing 2 mM MgCl2 were injected over the chip surface and the sensorgrams were recorded. As shown in Fig. 3, the sensorgrams detected little interaction between sGP Ibα and control chips or chips containing immobilized scM2. Significant interaction responses were detected for sGP Ibα with M2. Apparent equilibrium dissociation constant was estimated from the equilibrium resonance signal as a function of analyte (GP Ibα) concentration (kD ∼20 μM) and calculated by Scatchard analysis (kD = 17 μM). Similar results were obtained when purified sGP Ibα in running buffer containing 2 mM CaCl2 were injected over the chip surface (not depicted). Thus, BIAcore analysis revealed real-time, direct binding for bimolecular interactions between GP Ibα and M2.
GP Ibα Binding to “Triple Mutants” of the αMI Domain.
To begin localization of specific amino acid residues within the αMI domain involved in GP Ibα recognition, recombinant fragments containing wild-type αMI domain, R115–S340, wild-type αLI domain, P120–S330, and a mutant αMI domain were expressed as GST fusion proteins in Escherichia coli. The mutant αMI domain contained a swap of the P201–K217 segment, which we implicated in sGP Ibα binding to the corresponding residues of the αLI domain. The recombinant proteins were purified from the bacterial lysates on glutathione-Sepharose. A facile binding assay for quantifying sGP Ibα binding to the recombinant I domains was developed by measuring the binding of biotinylated sGP Ibα to the recombinant I domains immobilized onto 96-well plastic plates. As shown in Fig. 4, GP Ibα exhibited minimal reaction with αLI domain. Binding was observed and was similar with both human and mouse αMI domains. With the αMI domains, binding was dependent on the sGP Ibα concentration with 50% maximal binding observed at 50 nM sGP Ibα, which was used in subsequent experiments. As shown in Fig. 5 A, the interaction of the αMI domain “swap” mutant with biotinylated sGP Ibα was greatly diminished compared with wild-type αMI domain and similar to that of the αLI domain. Essentially, no specific binding of sGP Ibα to this mutant I domain was detected. This result is consistent with our BIAcore binding experiments showing a direct interaction between sGP Ibα and immobilized M2 peptide corresponding to this P201–K217 segment.
To begin to identify the individual residues within the P201–K217 segment that mediated sGP Ibα recognition, a series of six triple mutants were created. In each of these triple mutants, a set of three consecutive amino acids within the αMI domain was changed to the corresponding αL residues. If the αM and αL residues were the same, the amino acid was mutated to alanine. After the DNA sequence of each mutant I domain was confirmed, it was expressed in E. coli and purified on glutathione-Sepharose. When analyzed by SDS-PAGE, each mutant migrated as a single band of ∼52 kD (not depicted). sGP Ibα binding to each triple mutant was then assessed. The results in Fig. 5 A show the binding of each of the six triple mutants to 50 nM biotinylated sGP Ibα. Of the six triple mutants, three, H210–A212, T213–I215, and R216–K217, showed a significant reduction in sGP Ibα binding. Three mutants, P201–T203, Q204–L206, and G207–T209, did not impair binding to GP Ibα. These results, taken in light of the observation that mutating the entire region spanning P201–G207 abolished cell adhesion to sGP Ibα (Fig. 1), suggest that the inactivation of sGP Ibα binding requires alteration of more than one residue within the P201–G207 (i.e., no single residue within the triple mutants, P201–T203, Q204–L206, and G207–T209, decreases affinity detectably), or the conformation of the P201–G207 segment, which corresponds to a portion of a loop within the αMI domain, is necessary for binding, and, again, no single substitution alters the conformation of this region sufficiently to prevent binding.
GP Ibα Binding to “Single Point Mutants” of the αMI Domain.
Next, within the three triple mutants with reduced sGP Ibα binding, each of the three amino acids was mutated individually to the corresponding residue in αLI domain or in case of identical residues in two I domains, the amino acid was replaced with an alanine. After confirming the DNA sequences of these single mutants, each of the GST fusion proteins was purified. The mutant fusion protein carrying the K217Y substitution was insoluble. Therefore, K217 was mutated to Ala, and this αMI domain with a K217A substitution was readily purified, yielding a total of eight single mutants. The capacity of the eight single mutants to bind sGP Ibα is summarized in Fig. 5 B. Within each of the three triple mutants with reduced sGP Ibα recognition, only one of the three single mutants exhibited reduced sGP Ibα binding. These three single mutants showing reduced binding were T211A, T213G, and R216N.
Development of a Chimeric αLI Domain with GP Ibα Binding Activity.
Loss of GP Ibα binding function in these single mutants could reflect direct involvement of the specific residues in GP Ibα binding or conformational perturbation of the resulting αMI domain due to substitutions at these positions. A gain in function approach was used to distinguish between these possibilities by introducing the identified point mutations into the αLI domain. T211 is conserved in both αMI and αLI domains. Therefore, chimeric I domains containing either a single G213T substitution or two substitutions, G213T and N216R, in the αLI domain backbone were created. These mutant αLI domains were expressed as GST fusion proteins, purified, and their sGP Ibα binding properties were evaluated. The chimeric I domain harboring both the G213T and N216R mutations bound sGP Ibα with an affinity substantially greater than wild-type αLI domain and the I domain containing the single G213T mutation (Fig. 6). Indeed, the binding capability of double substituted chimeric I domain was comparable to the wild-type αMI domain. To confirm that these two amino acid residues are sufficient to impart sGP Ibα recognition to the mutated αLI domain, we performed additional BIAcore binding assays with immobilized αLI domain peptide (C201HVKHMLLTNTFGAINY217, termed C-L2) corresponding to the P201–K217 sequence within αMI domain and the double substituted mutant C201HVKHMLLTNTFTAIRY217 (C-muL2). Binding assays were also performed with a mutant M2 peptide containing T211A, T213G, and R216N substitutions (C201PITQLLGRTHAAGGINK217, termed C-muM2) corresponding to the three single mutants showing reduced binding in the purified mutant I domain binding assays (Fig. 5 B). The sensorgrams detected little interaction between 50 μM sGP Ibα and chips containing immobilized C-L2 (RU = 10) or C-muM2 (RU = 25). Significant interaction responses were detected for sGP Ibα with C-muL2 (RU = 224).
Role of the Identified Amino Acids in the Context of Intact αMβ2.
The role of T213 and R216 in the GP Ibα binding function of the intact receptor was investigated. The T213G and R216N substitutions were introduced into the cDNA for the αL subunit using site-directed mutagenesis and coexpressed with the cDNA for the β2 subunit in 293 cells. Wild-type αLβ2 and αMβ2 were also transiently expressed in these cells as controls. 48 h after transfection, the cells were detached from tissue culture plates, and receptor expression levels were evaluated by FACS®. αM expression was evaluated with OKM1, β2 expression with IB4, and αL expression with TS1/22. The expression levels of both the α and β subunits of the integrins were comparable (not depicted). Next, the function of the receptors was assessed by evaluating their adhesion to immobilized sGP Ibα. PMA, Mn2+, or their combination were used to activate the integrins on the cells. As shown in Fig. 7, mock-transfected cells or cells expressing wild-type αLβ2 showed little adhesion to sGP Ibα under all conditions. When αMβ2-expressing cells were stimulated with PMA, Mn2+, or their combination, their adhesion to sGP Ibα increased markedly compared with the nonstimulated cells. The chimeric αLβ2 cells adhered to sGP Ibα considerably better than the αLβ2 cells or mock-transfected cells. The adhesion of the chimeric αLβ2 cells approached that of the wild-type αMβ2 cells. We considered whether inserting these amino acid residues into αL might affect the activation state of chimeric αLβ2 such that the increase in binding might reflect allosteric changes in the integrin. Accordingly, we assessed the activation states of the chimeric integrin using MEM-83, an mAb that reacts with activated αLβ2 (33). In FACS® analyses, MEM-83 failed to react with either the αLβ2 or the chimeric αLβ2 transfectants in the unstimulated state (MFI = 14 and 10, respectively), but reacted well and equivalently with both transfectants upon stimulation with the combination of Mn2+ and PMA (MFI = 79 and 83, respectively). Thus, the activation states of both the αLβ2 and chimeric αLβ2 transfectants, with and without stimulation, were similar.
αMβ2 and GP Ibα Facilitate the Interaction between Leukocytes and Platelets.
Having previously reported that αMβ2 and GP Ibα facilitate the heterotypic interaction between leukocytes and platelets, we turned to examining the effect of M2 on neutrophil adhesion to platelets. Thioglycollate-elicited neutrophils were added to wells containing surface adherent platelets and adhesion was stimulated by the addition of PMA to each well. We verified by FACS® analysis that such PMA treatment up-regulated P-selectin expression 29.9-fold in platelets purified and prepared with PGE1 as described in Materials and Methods. Wild-type (Mac-1+/+) neutrophils bound to adherent platelets (Fig. 8). In contrast, Mac-1–deficient (Mac-1−/−) neutrophils demonstrated markedly reduced adhesion to platelets (percent wild-type adhesion = 18.2 ± 8.0) and adhesion of wild-type neutrophils was blocked by the rat anti–mouse αMβ2 mAb M1/70 (percent wild-type adhesion = 29.9 ± 19.8), indicating that under these conditions, neutrophil adhesion to platelets is largely αMβ2 dependent, although a complimentary role for P-selectin/PSGL-1 is likely operative. M2, but not a scrambled version, inhibited dose dependently (IC50 = 30 nM) wild-type neutrophil adhesion to platelets. Taken together, these observations indicate that under these experimental conditions, neutrophil adhesion to platelets is mediated primarily by the αMI domain sequence P201–K217.
M2 Peptide Abrogates the Firm Adhesion of THP-1 Cells under Flow.
To evaluate the potential for M2 to modulate the adhesion of blood cells under flow, we perfused THP-1 cells that express Mac-1 over coverslips coimmobilized with soluble P-selectin and GP Ibα using a parallel plate flow chamber system (0.75 dynes/cm2). The number of rolling and arrested cells was quantified on five random fields after 3 min of perfusion. THP-1 cells rolled and arrested on coverslips cocoated with P-selectin and sGP Ibα and the number of rolling (control vs. scM2, P > 0.05) or arrested (control vs. scM2, P > 0.05) cells was unaffected by scM2 (Fig. 9 A). In contrast, M2 peptide inhibited THP-1 cell arrest, thereby increasing the number of rolling cells visualized. The effect of M2 on cell adhesion was similar to treatment with LPM19c, an anti-CD11b mAb that blocks αMβ2-dependent binding to sGP Ibα, thereby confirming the involvement of αMβ2 in cell adhesion. Both cell rolling and arrest were abolished by treating cells with KPL-1, an anti–PSGL-1 mAb that blocks P-selectin binding. The effect of M2 on cell arrest was also quantified. Thus, on coverslips incubated with vehicle or scM2, 64 and 66% of THP-1 cells arrested, respectively (Fig. 9 B). After treatment with M2 peptide only 34% of cells arrested (P < 0.05). Similarly, after preincubation of THP-1 cells with LPM19C, only 33% of cells arrested (P < 0.05). KPL-1 antibody treatment abrogated almost all rolling and subsequent arrest (<10%; P < 0.01).
In this study, we have identified the P201–K217 segment, which spans an exposed loop and amphipathic α4 helix in the three-dimensional structure of the αMI domain, as the binding site for platelet GP Ibα. This conclusion is supported by the following data: (a) mutant cell lines in which the αMI domain segments P201–G207 and R208–K217 were switched to the homologous αLI domain segments failed to support adhesion to sGP Ibα, (b) mutation of amino acid residues within P201–K217, H210–A212, T213–I215, and R216–K217 resulted in the loss of the binding function of the recombinant αMI domains to biotinylated sGP Ibα, (c) synthetic peptides duplicating the P201–K217, but not scrambled versions, directly bound sGP Ibα and inhibited αMβ2-dependent adhesion to sGP Ibα and adherent platelets, and (d) grafting key amino acids within the P201–K217 sequence onto αL converted αLβ2 into a GP Ibα binding integrin.
By virtue of binding diverse ligands including, among others, fibrin(ogen) (34, 35), ICAM-1 (36), factor X (37), C3bi (34), high molecular weight kininogen (38), and heparin (4), αMβ2 regulates important leukocyte functions including adhesion, migration, coagulation, proteolysis, phagocytosis, oxidative burst, and signaling (30, 39–42). However, these ligands do not account for all of αMβ2's adhesive interactions. Although previous studies have shown that αMβ2 directly facilitates the recruitment of leukocytes at sites of platelet and fibrin deposition (5), the precise platelet counter receptors, including GP Ibα (11) and JAM-3 (43), have been elucidated only recently.
In this study, we have identified key elements of the binding site for GP Ibα within the αMI domain. The strategy to define the ligand binding site was based on the difference in the sGP Ibα binding properties of the αMI and αLI domains and entailed four complimentary approaches. In the first approach, a series of homologue-scanning mutants, used previously to map the binding regions for NIF, iC3b, and fibrinogen (16, 20, 23), were screened for adhesion to sGP Ibα. In these mutants, 16 segments at the hydrated surface of the αMI domain were replaced with the corresponding segments from the homologous αLI domain, which does not bind GP Ibα. 11 mutants lacked the ability to support adhesion sGP Ibα, and alteration of two other regions, αM(E178–T185) and αM(ΔE262G263), resulted in the partial loss of adhesive function. Thus, the initial insight provided by these mutant receptors indicated that the sGP Ibα binding interface within the αMI domain was composed of several nonlinear sequences.
The second approach entailed the use of synthetic peptides duplicating the sequences of the critical segments in the αM I domain. These analyses showed that two critical segments, αM(P201–G207) and αM(R208–K217), may contain amino acid residues that participate directly in binding GP Ibα because the peptide M2 that spanned P201–K217 bound sGP Ibα and inhibited αMβ2-dependent adhesion to sGP Ibα and adherent platelets. The negative results for other peptides (M1, M3–M5, and M7) synthesized to correspond to other negative mutants (Fig. 1 and Table I) do not exclude a role for other αMI domain segments in binding function. These segments may play an accessory role in ligand binding or the short peptides may simply not assume the appropriate conformation for recognition by the ligand. Finally, segments implicated in the binding of sGP Ibα by the negative mutants, other than P201–K217, may reflect interference by αLI domain residues rather than residues that actually participate in binding. It is also possible that the activation of αMβ2, rather than ligand binding per se, was affected adversely by the introduction of these αL segments within αMI domain. Because these same mutants have been used in previous studies (17, 20, 23) to analyze interaction of other activation-dependent ligands with αMβ2, such effects on activation would need to specifically perturb GP Ibα recognition.
To obtain direct evidence that the αM(P201–K217) sequence constitutes the functional binding site for the GP Ibα, we turned to a third approach using site-directed mutagenesis of the αMI domain. Binding experiments with purified sGP Ibα and GST-I domain mutants provided the independent confirmation that the P201–K217 segment is important for sGP Ibα binding because mutations of these residues resulted in significant loss of sGP Ibα binding. These experiments also served to narrow further the binding region to H210–K217 and subsequently identified three single mutants showing reduced binding to sGP Ibα (T211A, T213G, and R216N). Of these, T211 in αMI domain is conserved in the αLI domain. Thus, it was the conversion of the residue to A that perturbed GP Ibα binding, suggesting that this substitution exerts a negative influence on the conformation of the αMI domain that is required for GP Ibα recognition, rather than participating directly in ligand contact. The positioning of this residue on the interior of the α4-helix and facing the central core of the αMI domain, according to the crystal structures (8, 9), supports this interpretation. In contrast, T213 and R216 are appropriately positioned, including when the recent crystal structure of the GP Ibα/vWF A domain (44) is used as a template.
In the fourth approach, the two amino acids (T213G and R216N substitutions) within the αM(P201–K217) were grafted into the corresponding positions of the αLI domain in the context of intact αLβ2. As demonstrated in Figs. 6 and 7, this manipulation imparted GP Ibα binding capacity to the chimeric molecule. Thus, the role of amino acids within the αM(P201–K217) sequence in GP Ibα binding, which initially was inferred from the loss in function experiments, was verified by the gain in function approach. Taken together, the four approaches substantiated independently the role of αM(P201–K217) in GP Ibα binding and provided evidence that two to three critical residues participate in ligand docking.
To date, several lines of evidence have emphasized that multiple ligands share overlapping binding sites within αMβ2, including the fact that one ligand (e.g., NIF) is capable of blocking the interaction of multiple ligands (C3bi, ICAM-1, fibrinogen) with the receptor (15, 45). However, although the binding sites for these ligands might be overlapping, they need not be identical (16, 17). Ustinov and Plow (22) have proposed a mosaic model in which many of the same loops and helices of the αMI domain, on or near its MIDAS face, may engage ligands, but different amino acid residues within these structures may contact the ligands. Direct support for this mosaic model is seen within the K245–R261 segment of the αMI domain, which has been implicated in both NIF and fibrinogen recognition (23). Key contact amino acids in this loop for fibrinogen binding are F246, D254, and P257, whereas Y252 and E258 are involved in NIF binding. This same αMI domain segment is also involved in C3bi recognition by αMβ2 (17).
Under the experimental conditions used in this study, which assayed the adhesion of activated neutrophils to surface-adherent platelets after vigorous washing, the predominant interaction between neutrophils and platelets appeared to be mediated by αMβ2 binding to sGP Ibα, based on the ability of M2 to inhibit >80% of neutrophil adhesion. The enhanced potency of M2 with respect to inhibiting neutrophil adhesion to platelets (IC50 = 30 nM) compared with BIAcore (kD = 17 μM) is possibly secondary to the fact that leukocytes express αMβ2 and platelets express GP Ibα in their native conformations. In contrast, BIAcore experiments used sGPIbα with immobilized M2 peptide.
Our data do not rule out the possibility of additional platelet surface receptors for αMβ2. Other potential αMβ2 ligands present on the platelet membrane include fibrinogen (bound to GP IIb-IIIa; 34, 35), ICAM-2 (46), high molecular weight kininogen (38), and JAM-3 (43). However, a leukocyte–platelet interaction mediated by fibrinogen bridging between αMβ2 and GP IIb/IIIa has been discounted by Ostrovsky et al. (47), who found that neither RGDS peptides nor the replacement of normal platelets with thrombasthenic platelets (i.e., lacking GP IIb/IIIa) affected the accumulation of the leukocytes on platelets. Although αMβ2 binds ICAM-1, this receptor is not found on platelets. Platelets express a related receptor, ICAM-2 (48), but Diacovo et al. (5) have shown that ICAM-2 blockade has no effect on the firm adhesion of neutrophils on monolayers of activated platelets under flow. Santoso et al. (43) have reported recently that αMβ2 may also bind to platelet JAM-3, cooperating with GP Ibα to mediate neutrophil–platelet adhesive contacts (43).
The present observations also suggest a possible target for therapeutic intervention. In particular, the specificity of M2 inhibitory action toward GP Ibα (i.e., noninhibitory toward fibrinogen) suggests that it might be possible to prevent leukocyte attachment to platelets by targeting GP Ibα without inhibiting other αMβ2 functions. Our recent observations have identified αMβ2 as a molecular determinant of neointimal thickening after experimental arterial injury that produces endothelial denudation and platelet deposition. We found that antibody-mediated blockade (49) or selective absence (50) of αMβ2 impaired transplatelet leukocyte migration into the vessel wall, diminishing medial leukocyte accumulation and neointimal thickening after experimental angioplasty or endovascular stent implantation. Therefore, this study identifying the precise binding site responsible for αMβ2–GP Ibα interaction might provide a molecular strategy for disrupting leukocyte–platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.
This work was supported in part by grants from the National Institutes of Health (HL53993, HL36028, and HL65090 to F.W. Luscinskas, HL65967 and HL64796 to J.A. Lopez, HL66197 to E.F. Plow, and HL57506 and HL60942 to D.I. Simon). R. Ehlers received a post-doctoral research fellowship from Society for Thrombosis and Hemostasis Research, Germany. V. Ustinov received a post-doctoral research fellowship from The Ohio Valley Affiliate, American Heart Association (0120394B). R.M. Rao is an ARC Copeman Traveling Research Fellow.
Note added in proof. Hoffmeister et al. (Hoffmeister, K.M., E.C. Josefsson, N.A. Isaac, H. Clausen, J.H. Hartwig, and T.P. Stossel. 2003. Glycosylation restores survival of chilled blood platelets. Science. 301:1531–1534.) have recently reported that chilled platelets are cleared by hepatic macrophage αMβ2 receptors via an interaction between platelet GP Iβα and a non–I-domain, lectin-binding site within aMβ2.
R. Ehlers and V. Ustinov contributed equally to this work.
Abbreviations used in this paper: BCECF AM, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester; GP, glycoprotein; GST, glutathione S-transferase; ICAM, intercellular adhesion molecule; MFI, mean fluorescence intensity; NIF, neutrophil inhibitory factor; RU, resonance unit; sGP Ibα, soluble extracellular region of GP Ibα (i.e., glycocalicin); vWf, von Willebrand factor.