© Rockefeller University Press,
0022-1007/2003/10/1077 $5.00
The Journal of Experimental Medicine, Volume 198, Number 7, 1077-1088
Targeting PlateletLeukocyte Interactions
:
Identification of the Integrin Mac-1 Binding Site for the Platelet Counter Receptor Glycoprotein Ib
Raila Ehlers1,
Valentin Ustinov3,
Zhiping Chen1,
Xiaobin Zhang1,
Ravi Rao2,
F. William Luscinskas2,
Jose Lopez4,
Edward Plow3 and
Daniel I. Simon1
1 Cardiovascular Division, Brigham and Women's Hospital, Boston, MA 02115
2 Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115
3 The Cleveland Clinic Foundation, Cleveland, OH 44195
4 Thrombosis Research Section, Departments of Medicine and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030
Address correspondence to Daniel I. Simon, Brigham and Women's Hospital, Cardiovascular Division, 75 Francis Street, Tower 3, Boston, MA 02115. Phone: (617) 732-5867; Fax: (617) 732-5132; email: dsimon{at}rics.bwh.harvard.edu
 |
Abstract
|
---|
The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are dependent on the interaction of the leukocyte integrin Mac-1 (
Mß2, CD11b/CD18) and the platelet counter receptor glycoprotein (GP) Ib
. Previous studies have established a central role for the I domain, a stretch of
200 amino acids within the
M subunit, in the binding of GP Ib
. This study was undertaken to establish the molecular basis of GP Ib
recognition by
Mß2. The P201K217 sequence, which spans an exposed loop and amphipathic
4 helix in the three-dimensional structure of the
MI domain, was identified as the binding site for GP Ib
. Mutant cell lines in which the
MI domain segments P201G207 and R208K217 were switched to the homologous, but non-GP Ib
binding,
L domain segments failed to support adhesion to GP Ib
. Mutation of amino acid residues within P201K217, H210A212, T213I215, and R216K217 resulted in the loss of the binding function of the recombinant
MI domains to GP Ib
. Synthetic peptides duplicating the P201K217, but not scrambled versions, directly bound GP Ib
and inhibited
Mß2-dependent adhesion to GP Ib
and adherent platelets. Finally, grafting critical amino acids within the P201K217 sequence onto
L, converted
Lß2 into a GP Ib
binding integrin. Thus, the P201K217 sequence within the
MI domain is necessary and sufficient for GP Ib
binding. These observations provide a molecular target for disrupting leukocyteplatelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.
Key Words: inflammation leukocytes platelets adhesion receptors
 |
Introduction
|
---|
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 ß2 integrin family, LFA-1 (
Lß 2, CD11a/CD18), Mac-1 (
Mß2, CD11b/CD18), and p150,95 (
Mß2, 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
Mß2 (5).
Our laboratory has focused on identifying the platelet counter receptor for
Mß2. Evaluation of the structural features of integrins provides insight into candidate platelet counter receptors for
Mß2. Integrins are heterodimeric proteins composed of one
and one ß subunit. A subset of integrin
subunits, including
M, contains an inserted domain (I domain) of
200 amino acids that is implicated in ligand binding (79) 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
MI domain, we hypothesized that GP Ib
might also be able to bind
Mß2 and reported that GP Ib
is indeed a constitutively expressed counter receptor for
Mß2 (11). Furthermore, under the conditions used in these studies, the predominant interaction between neutrophils and platelets appeared to be between
Mß2 and GP Ib
(11).
The
MI domain contributes broadly to the recognition of ligands by
Mß2 (12) and specifically to the binding of GP Ib
(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 Ib
. 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
MI domain have been mapped extensively (1823), the recognition site for GP Ib
is unknown.
In this study, we have localized the binding site for GP Ib
within the
MI domain. The strategy developed was based on the differences in the binding of GP Ib
to the
MI and
LI 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 Ib
was localized within the segment
M(P201K217). The grafting of two amino acids within this segment into the
LI domain converted it to a GP Ib
binding protein. Thus, a small segment that has a defined structure within the
MI domain is necessary and sufficient for GP Ib
binding.
 |
Materials and Methods
|
---|
Materials.
The soluble extracellular region of GP Ib
(sGP Ib
; 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
MI domain (provided by K. Pulford, Radcliffe, Oxford, United Kingdom; 12); OKM1, directed to the
M subunit of human Mac-1 (American Type Culture Collection [ATCC]); M1/70, directed to the
M subunit mouse
Mß2 (ATCC; 24); CBRM1/5, an activation-specific
M reporter antibody (provided by T. Springer, Harvard Medical School, Boston, MA; 25); TS1/22, directed to the
L subunit of
Lß2 and capable of blocking ICAM-1 binding (ATCC); MEM-83, an activation-specific
L reporter antibody (Caltag); and IB4, a blocking mAb directed to the ß2 subunit (ATCC). The stimulating CD18 mAb KIM127 (26) was provided by M. Robinson (Celltech Ltd., Slough, United Kingdom). mAb24, a ß2 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.
THP-1 monocytic cells (ATCC) were maintained and differentiated with 1 ng/ml TGF-ß1 and 50 nM 1,25-(OH)2 vitamin D3, as previously described (28). 293 cells expressing human
Lß2,
Mß2, or mutant
Mß2 receptors were established and maintained as previously described (16, 20, 23, 29).
Segment Switches by Site-directed Mutagenesis.
To systematically define the GP Ib
binding site in
M, a homologue-scanning mutagenesis strategy was implemented (20). Accordingly, guided by the crystal structure (8, 9), the hydrated surface of the
MI domain was replaced with sequences of the
LI domain in segments of 711 amino acids. To apply this approach to the
MI domain (
200 amino acids), 16 segments were switched (20, 23). Site-directed mutagenesis of the
MI 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
M subunit cDNA.
Transient Transfection.
The expression vector pcDNA3.1 (Invitrogen) was used for cloning
M,
L, and ß2 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.
Flow Cytometry.
FACS® analyses were performed to assess the expression of wild-type and mutated forms of
Mß2 and
Lß2 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-1deficient (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).
Adhesion Assays.
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 Ib
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 ß2-stimulating mAb KIM 127. Plates were washed and adhesion was quantified by measuring the fluorescence of BCECF AMloaded cells using a Cytofluor II fluorescence microplate reader (PerSeptive Biosystems). The effect of anti-
M mAb on adhesion was assessed by preincubating cells with 10 µg/ml LPM19c. The effect of peptides M1M8 on adhesion was investigated by incubating the indicated peptide with sGP Ib
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
M peptides on leukocyte adhesion to platelets was examined by preincubating surface-adherent platelets with peptide (11,000 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
MI domain (675 nucleotides, R115S340),
LI domain (630 nucleotides, P120S330), and mouse
MI domain (675 nucleotides, L115S340) 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
L 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 Ib
Binding to the I Domains.
To test the interaction of the wild-type
M,
L, and chimeric I domains with biotinylated GP Ib
, 96-well plates (Immulon 4BX; Dynex Technologies Inc.) were coated with the I domains at 50 µg/ml and blocked with 2% BSA. sGP Ib
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 Ib
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 proteinprotein 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 Ib
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 Ib
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
Mß2 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.
Mß2 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 x 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.
Statistics.
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.
 |
Results
|
---|
Binding of GP Ib
to Mutant Cell Lines.
Our previous report indicated that the
MI domain serves as a recognition site for GP Ib
(11). The adhesion of
Mß2-bearing cells to sGP Ib
was inhibited by LPM19c, an mAb that binds to the
MI domain. To establish definitively that the
MI domain serves as a recognition site for GP Ib
, we transfected 293 cells with wild-type
Mß2,
Lß2, or a chimeric
Lß2 receptor that contained the I domain of
Mß2 (i.e.,
L(I
M)ß2-transfected 293 cells).
Lß2-transfected 293 cells did not adhere to sGP Ib
. In contrast,
L(I
M)ß2-transfected 293 cells adhered robustly to sGP Ib
in a manner similar to
Mß2-transfected 293 cells, indicating that the
MI domain is required for adhesion to sGP Ib
. These data suggest that although
Mß2 and
Lß2 are highly homologous integrins, the lack of binding of sGP Ib
to
Lß2 might be the result of sequence and/or structural differences.
As the first step to define the binding site for GP Ib
within the
MI domain, mutant cell lines, each expressing a mutant
Mß2 in which a short
MI domain sequence, corresponding to a structural unit in the crystal structure (8, 9), was replaced for the corresponding region of the
LI domain, were tested for their adhesion to immobilized sGP Ib
. 16 segments were switched from their original
MI domain to their counterparts in
L (16, 20, 23). These segment swaps are placed throughout the P147Q314 region of the
MI domain, D132A318, 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 Ib
are summarized in Fig. 1. The data are expressed as the percent adhesion of the wild-type
Mß2-expressing cells to sGP Ib
. Substitutions for the following regions of
MI domain abrogated adhesion:
M(P147R152),
M(M153T159),
M(E162L170),
M(P201G207),
M(R208K217),
M(K245FG247),
M (
D248Y252),
M(E253R261),
M(D273K279),
M(R281I287), and
M(F297T307). These cell lines form a group of negative mutants. As the essential control, the
Lß2-expressing cells adhered poorly to sGP Ib
, consistent with lack of interaction of
Lß2 with sGP Ib
.

View larger version (16K):
[in this window]
[in a new window]
|
Figure 1. Binding of GP Ib to mutant cell lines. Adhesion of 105 Mß2-, Lß2-, and mutant Mß2-expressing 293 cells to sGP Ib coated wells. Adhesion was promoted by the addition of 5 µg/ml of the stimulating mAb KIM 127 and quantified by measuring the fluorescence of BCECF AMloaded cells. Data are expressed as percent wild-type Mß2 adhesion (mean ± SD of three to five independent experiments). A series of peptides (M1M8) corresponding to the wild-type MI domain sequences were synthesized and tested for their ability to interact with sGP Ib (Table I). Because the sequences within several of the negative mutants were contiguous, linear peptides spanning these sequences were synthesized: P147T159 (M1), P201K217 (M2), K245R261 (M3), D273I287 (M4), F297T307 (M5), Q190S197 (M6), E162L170 (M7), and E178T185 (M8). All peptides correspond to negative mutant sequences except M6 and M8, which correspond to intermediate and positive mutant sequences, respectively.
| |
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
M(P201G207) mutant was 1.5-fold higher than that of the cells expressing the wild-type
Mß2 cells, but adhesion was abrogated completely. Surface expression of negative mutants,
M(P147R152),
M(R208K217),
M(E253R261), and
M(D273K279) was very similar to that of the wild-type receptor. Adhesion of
M(E178T185) and
E262G263 was partially affected. The maximal level of adhesion to sGP Ib
reached 69 and 64%, respectively, of wild-type
Mß2 cells. These two receptors were classified as intermediate mutants.
The following mutants supported the same or higher levels of adhesion to sGP Ib
compared with the cells expressing the wild-type
Mß2 receptor:
M(Q190S197),
M(K231NAF234),
M(E262G263), and
M(Q309E314). The cell lines exhibiting adhesion similar to or greater than the wild-type
Mß2 cells were identified as positive mutants. Two of these positive mutants,
M(Q190S197) and
M(K231NAF234), bound sGP Ib
better than wild-type
Mß2. Previous analyses using these mutants showed that they bound neither an
Mß2 activation-independent ligand, NIF (20), nor
Mß2 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 Ib
recognition. In further studies, we found that binding of the ß2 activationspecific reporter mAb 24 was similar for wild-type and the
M (Q190S197) and
M(K231NAF234) mutants. In contrast, increased binding of the
M activationspecific reporter CBRM1/5 to
M(K231NAF234) compared with wild-type
Mß2 and
M(Q190S197) suggests possible conformation change of the
M(K231NAF234) mutant (not depicted). Together, these observations suggest that the activation states of
Mß2 may reflect the reporter ligand or mAb used, and these two mutants might be functionally activated with respect to sGP Ib
recognition.
Interaction of GP Ib
with the
MI Domain Peptides.
In subsequent analyses, we focused on the 11 negative mutants. A series of peptides (M1M8) corresponding to the wild-type
MI domain sequences were synthesized and tested for their ability to interact with sGP Ib
. Because the sequences within several of the negative mutants were contiguous, the following linear peptides spanning these sequences were synthesized: P147T159 (designated M1), P201K217 (M2), K245R261 (M3), D273I287 (M4), F297T307 (M5), Q190S197 (M6), E162L170 (M7), and E178T185 (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
MI domain peptides on
Mß2-dependent adhesion to sGP Ib
or fibrinogen (Table I). 50 µM peptides were preincubated in ligand-coated wells,
Mß2-expressing 293 cells were added, and adhesion was stimulated with KIM 127. Mac-1 293 cells adhered to sGP Ib
and fibrinogen, and this adhesion was blocked by the anti-CD11b mAb LPM19c, indicating that adhesion is predominantly
Mß2 dependent (Table I). Peptide M2 inhibited adhesion to sGP Ib
(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
Mß2-expressing 293 cell adhesion to sGP Ib
or fibrinogen. M2, but not a scrambled version (scM2), blocked
Mß2-dependent adhesion to sGP Ib
in a dose-dependent manner (IC50 = 20 µM; Fig. 2). Taken together, these observations suggest that the
MI domain sequence corresponding to M2 (P201K217), which spans an exposed loop and amphipathic
4 helix in the three-dimensional structure of the
MI domain, contributes to
Mß2 binding of GP Ib
.
Real-Time Detection of ProteinProtein Interaction by BIAcore Analyses Reveals Specific, Direct Binding of GP Ib
to M2 Peptide.
To examine direct interactions between GP Ib
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
Mß2-dependent adhesion to sGP Ib
, verifying that cysteine addition did not adversely affect peptide structure (Table I). Increasing concentrations of purified sGP Ib
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 Ib
and control chips or chips containing immobilized scM2. Significant interaction responses were detected for sGP Ib
with M2. Apparent equilibrium dissociation constant was estimated from the equilibrium resonance signal as a function of analyte (GP Ib
) concentration (kD
20 µM) and calculated by Scatchard analysis (kD = 17 µM). Similar results were obtained when purified sGP Ib
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 Ib
and M2.
GP Ib
Binding to "Triple Mutants" of the
MI Domain.
To begin localization of specific amino acid residues within the
MI domain involved in GP Ib
recognition, recombinant fragments containing wild-type
MI domain, R115S340, wild-type
LI domain, P120S330, and a mutant
MI domain were expressed as GST fusion proteins in Escherichia coli. The mutant
MI domain contained a swap of the P201K217 segment, which we implicated in sGP Ib
binding to the corresponding residues of the
LI domain. The recombinant proteins were purified from the bacterial lysates on glutathione-Sepharose. A facile binding assay for quantifying sGP Ib
binding to the recombinant I domains was developed by measuring the binding of biotinylated sGP Ib
to the recombinant I domains immobilized onto 96-well plastic plates. As shown in Fig. 4, GP Ib
exhibited minimal reaction with
LI domain. Binding was observed and was similar with both human and mouse
MI domains. With the
MI domains, binding was dependent on the sGP Ib
concentration with 50% maximal binding observed at 50 nM sGP Ib
, which was used in subsequent experiments. As shown in Fig. 5 A, the interaction of the
MI domain "swap" mutant with biotinylated sGP Ib
was greatly diminished compared with wild-type
MI domain and similar to that of the
LI domain. Essentially, no specific binding of sGP Ib
to this mutant I domain was detected. This result is consistent with our BIAcore binding experiments showing a direct interaction between sGP Ib
and immobilized M2 peptide corresponding to this P201K217 segment.
To begin to identify the individual residues within the P201K217 segment that mediated sGP Ib
recognition, a series of six triple mutants were created. In each of these triple mutants, a set of three consecutive amino acids within the
MI domain was changed to the corresponding
L residues. If the
M and
L 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 Ib
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 Ib
. Of the six triple mutants, three, H210A212, T213I215, and R216K217, showed a significant reduction in sGP Ib
binding. Three mutants, P201T203, Q204L206, and G207T209, did not impair binding to GP Ib
. These results, taken in light of the observation that mutating the entire region spanning P201G207 abolished cell adhesion to sGP Ib
(Fig. 1), suggest that the inactivation of sGP Ib
binding requires alteration of more than one residue within the P201G207 (i.e., no single residue within the triple mutants, P201T203, Q204L206, and G207T209, decreases affinity detectably), or the conformation of the P201G207 segment, which corresponds to a portion of a loop within the
MI domain, is necessary for binding, and, again, no single substitution alters the conformation of this region sufficiently to prevent binding.
GP Ib
Binding to "Single Point Mutants" of the
MI Domain.
Next, within the three triple mutants with reduced sGP Ib
binding, each of the three amino acids was mutated individually to the corresponding residue in
LI 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
MI 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 Ib
is summarized in Fig. 5 B. Within each of the three triple mutants with reduced sGP Ib
recognition, only one of the three single mutants exhibited reduced sGP Ib
binding. These three single mutants showing reduced binding were T211A, T213G, and R216N.
Development of a Chimeric
LI Domain with GP Ib
Binding Activity.
Loss of GP Ib
binding function in these single mutants could reflect direct involvement of the specific residues in GP Ib
binding or conformational perturbation of the resulting
MI 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
LI domain. T211 is conserved in both
MI and
LI domains. Therefore, chimeric I domains containing either a single G213T substitution or two substitutions, G213T and N216R, in the
LI domain backbone were created. These mutant
LI domains were expressed as GST fusion proteins, purified, and their sGP Ib
binding properties were evaluated. The chimeric I domain harboring both the G213T and N216R mutations bound sGP Ib
with an affinity substantially greater than wild-type
LI 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
MI domain. To confirm that these two amino acid residues are sufficient to impart sGP Ib
recognition to the mutated
LI domain, we performed additional BIAcore binding assays with immobilized
LI domain peptide (C201HVKHMLLTNTFGAINY217, termed C-L2) corresponding to the P201K217 sequence within
MI 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 Ib
and chips containing immobilized C-L2 (RU = 10) or C-muM2 (RU = 25). Significant interaction responses were detected for sGP Ib
with C-muL2 (RU = 224).
Role of the Identified Amino Acids in the Context of Intact
Mß2.
The role of T213 and R216 in the GP Ib
binding function of the intact receptor was investigated. The T213G and R216N substitutions were introduced into the cDNA for the
L subunit using site-directed mutagenesis and coexpressed with the cDNA for the ß2 subunit in 293 cells. Wild-type
Lß2 and
Mß2 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®.
M expression was evaluated with OKM1, ß2 expression with IB4, and
L expression with TS1/22. The expression levels of both the
and ß subunits of the integrins were comparable (not depicted). Next, the function of the receptors was assessed by evaluating their adhesion to immobilized sGP Ib
. 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
Lß2 showed little adhesion to sGP Ib
under all conditions. When
Mß2-expressing cells were stimulated with PMA, Mn2+, or their combination, their adhesion to sGP Ib
increased markedly compared with the nonstimulated cells. The chimeric
Lß2 cells adhered to sGP Ib
considerably better than the
Lß2 cells or mock-transfected cells. The adhesion of the chimeric
Lß2 cells approached that of the wild-type
Mß2 cells. We considered whether inserting these amino acid residues into
L might affect the activation state of chimeric
Lß2 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
Lß2 (33). In FACS® analyses, MEM-83 failed to react with either the
Lß2 or the chimeric
Lß2 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
Lß2 and chimeric
Lß2 transfectants, with and without stimulation, were similar.
Mß2 and GP Ib
Facilitate the Interaction between Leukocytes and Platelets.
Having previously reported that
Mß2 and GP Ib
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-1deficient (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 antimouse
Mß2 mAb M1/70 (percent wild-type adhesion = 29.9 ± 19.8), indicating that under these conditions, neutrophil adhesion to platelets is largely
Mß2 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
MI domain sequence P201K217.
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 Ib
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 Ib
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
Mß2-dependent binding to sGP Ib
, thereby confirming the involvement of
Mß2 in cell adhesion. Both cell rolling and arrest were abolished by treating cells with KPL-1, an antiPSGL-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).
 |
Discussion
|
---|
In this study, we have identified the P201K217 segment, which spans an exposed loop and amphipathic
4 helix in the three-dimensional structure of the
MI domain, as the binding site for platelet GP Ib
. This conclusion is supported by the following data: (a) mutant cell lines in which the
MI domain segments P201G207 and R208K217 were switched to the homologous
LI domain segments failed to support adhesion to sGP Ib
, (b) mutation of amino acid residues within P201K217, H210A212, T213I215, and R216K217 resulted in the loss of the binding function of the recombinant
MI domains to biotinylated sGP Ib
, (c) synthetic peptides duplicating the P201K217, but not scrambled versions, directly bound sGP Ib
and inhibited
Mß2-dependent adhesion to sGP Ib
and adherent platelets, and (d) grafting key amino acids within the P201K217 sequence onto
L converted
Lß2 into a GP Ib
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),
Mß2 regulates important leukocyte functions including adhesion, migration, coagulation, proteolysis, phagocytosis, oxidative burst, and signaling (30, 3942). However, these ligands do not account for all of
Mß2's adhesive interactions. Although previous studies have shown that
Mß2 directly facilitates the recruitment of leukocytes at sites of platelet and fibrin deposition (5), the precise platelet counter receptors, including GP Ib
(11) and JAM-3 (43), have been elucidated only recently.
In this study, we have identified key elements of the binding site for GP Ib
within the
MI domain. The strategy to define the ligand binding site was based on the difference in the sGP Ib
binding properties of the
MI and
LI 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 Ib
. In these mutants, 16 segments at the hydrated surface of the
MI domain were replaced with the corresponding segments from the homologous
LI domain, which does not bind GP Ib
. 11 mutants lacked the ability to support adhesion sGP Ib
, and alteration of two other regions,
M(E178T185) and
M(
E262G263), resulted in the partial loss of adhesive function. Thus, the initial insight provided by these mutant receptors indicated that the sGP Ib
binding interface within the
MI 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
M I domain. These analyses showed that two critical segments,
M(P201G207) and
M(R208K217), may contain amino acid residues that participate directly in binding GP Ib
because the peptide M2 that spanned P201K217 bound sGP Ib
and inhibited
Mß2-dependent adhesion to sGP Ib
and adherent platelets. The negative results for other peptides (M1, M3M5, and M7) synthesized to correspond to other negative mutants (Fig. 1 and Table I) do not exclude a role for other
MI 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 Ib
by the negative mutants, other than P201K217, may reflect interference by
LI domain residues rather than residues that actually participate in binding. It is also possible that the activation of
Mß2, rather than ligand binding per se, was affected adversely by the introduction of these
L segments within
MI domain. Because these same mutants have been used in previous studies (17, 20, 23) to analyze interaction of other activation-dependent ligands with
Mß2, such effects on activation would need to specifically perturb GP Ib
recognition.
To obtain direct evidence that the
M(P201K217) sequence constitutes the functional binding site for the GP Ib
, we turned to a third approach using site-directed mutagenesis of the
MI domain. Binding experiments with purified sGP Ib
and GST-I domain mutants provided the independent confirmation that the P201K217 segment is important for sGP Ib
binding because mutations of these residues resulted in significant loss of sGP Ib
binding. These experiments also served to narrow further the binding region to H210K217 and subsequently identified three single mutants showing reduced binding to sGP Ib
(T211A, T213G, and R216N). Of these, T211 in
MI domain is conserved in the
LI domain. Thus, it was the conversion of the residue to A that perturbed GP Ib
binding, suggesting that this substitution exerts a negative influence on the conformation of the
MI domain that is required for GP Ib
recognition, rather than participating directly in ligand contact. The positioning of this residue on the interior of the
4-helix and facing the central core of the
MI 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 Ib
/vWF A domain (44) is used as a template.
In the fourth approach, the two amino acids (T213G and R216N substitutions) within the
M(P201K217) were grafted into the corresponding positions of the
LI domain in the context of intact
Lß2. As demonstrated in Figs. 6 and 7, this manipulation imparted GP Ib
binding capacity to the chimeric molecule. Thus, the role of amino acids within the
M(P201K217) sequence in GP Ib
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
M(P201K217) in GP Ib
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
Mß2, 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
MI 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 K245R261 segment of the
MI 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
MI domain segment is also involved in C3bi recognition by
Mß2 (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
Mß2 binding to sGP Ib
, 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
Mß2 and platelets express GP Ib
in their native conformations. In contrast, BIAcore experiments used sGPIb
with immobilized M2 peptide.
Our data do not rule out the possibility of additional platelet surface receptors for
Mß2. Other potential
Mß2 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 leukocyteplatelet interaction mediated by fibrinogen bridging between
Mß2 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
Mß2 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
Mß2 may also bind to platelet JAM-3, cooperating with GP Ib
to mediate neutrophilplatelet adhesive contacts (43).
The present observations also suggest a possible target for therapeutic intervention. In particular, the specificity of M2 inhibitory action toward GP Ib
(i.e., noninhibitory toward fibrinogen) suggests that it might be possible to prevent leukocyte attachment to platelets by targeting GP Ib
without inhibiting other
Mß2 functions. Our recent observations have identified
Mß2 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
Mß2 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
Mß2GP Ib
interaction might provide a molecular strategy for disrupting leukocyteplatelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.
 |
Acknowledgments
|
---|
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. 301:15311534.) have recently reported that chilled platelets are cleared by hepatic macrophage
Mß2 receptors via an interaction between platelet GP Iß
and a nonI-domain, lectin-binding site within aMß2.
Submitted: December 20, 2002
Revised: August 6, 2003
Accepted: August 12, 2003
 |
Footnotes
|
---|
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 Ib
, soluble extracellular region of GP Ib
(i.e., glycocalicin); vWf, von Willebrand factor.
 |
References
|
---|
- Springer, T.A. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: a multistep paradigm. Cell. 76:301314.[Medline]
- Smith, C.W., S.D. Marlin, R. Rothlein, C. Toman, and D.C. Anderson. 1989. Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro. J. Clin. Invest. 83:20082017.[Medline]
- Languino, L.R., A. Duperray, K.J. Joganic, M. Fornaro, G.B. Thornton, and D.C. Altieri. 1995. Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition. Proc. Natl. Acad. Sci. USA. 92:15051509.[Abstract]
- Diamond, M.S., R. Alon, C.A. Parkos, M.T. Quinn, and T.A. Springer. 1995. Heparin is an adhesive ligand for the leukocyte integrin Mac-1 (CD11b/CD18). J. Cell Biol. 130:14731482.[Abstract]
- Diacovo, T.G., S.J. Roth, J.M. Buccola, D.F. Bainton, and T.A. Springer. 1996. Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the beta 2-integrin CD11b/CD18. Blood. 88:146157.[Abstract/Free Full Text]
- McEver, R.P., and R.D. Cummings. 1997. Role of PSGL-1 binding to selectins in leukocyte recruitment. J. Clin. Invest. 100:S97S103.[Medline]
- Diamond, M.S., J. Garcia-Aguilar, J.K. Bickford, A.L. Corbi, and T.A. Springer. 1993. The I domain is a major recognition site on the leukocyte integrin Mac-1 (CD11b/CD18) for four distinct adhesion ligands. J. Cell Biol. 120:10311043.[Abstract]
- Lee, J.O., P. Rieu, M.A. Arnaout, and R. Liddington. 1995. Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell. 80:631638.[Medline]
- Qu, A., and D.J. Leahy. 1995. Crystal structure of the I-domain from the CD11a/CD18 (LFA-1, alpha L beta 2) integrin. Proc. Natl. Acad. Sci. USA 92:10277-10281.[Abstract]
- Sadler, J.E., B.B. Shelton-Inloes, J.M. Sorace, J.M. Harlan, K. Titanti, and E.W. Davie. 1985. Cloning and characterization of two cDNAs coding for human von Willebrand factor. Proc. Natl. Acad. Sci. USA. 82:63946398.[Abstract]
- Simon, D.I., Z. Chen, H. Xu, C.Q. Li, J. Dong, L.V. McIntire, C.M. Ballantyne, L. Zhang, M.I. Furman, M.C. Berndt, et al. 2000. Platelet glycoprotein Ib
is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18). J. Exp. Med. 192:193204.[Abstract/Free Full Text]
- Diamond, M., J. Garcia-Aguiliar, J. Bickford, A. Corbi, and T. Springer. 1993. The I domain is a major recognition site on the leukocyte integrin Mac-1 (CD11b/CD18) for four distinct ligands. J. Cell Biol. 120:10311043.[Abstract]
- Zhou, L., D.H. Lee, J. Plescia, C.Y. Lau, and D.C. Altieri. 1994. Differential ligand binding specificities of recombinant CD11b/CD18 integrin I-domain. J. Biol. Chem. 269:1707517079.[Abstract/Free Full Text]
- Ueda, T., P. Rieu, J. Brayer, and M.A. Arnaout. 1994. Identification of the complement iC3b binding site in the beta 2 integrin CR3 (CD11b/CD18). Proc. Natl. Acad. Sci. USA. 91:1068010684.[Abstract/Free Full Text]
- Rieu, P., T. Ueda, I. Haruta, C.P. Sharma, and M.A. Arnaout. 1994. The A-domain of beta 2 integrin CR3 (CD11b/CD18) is a receptor for the hookworm-derived neutrophil adhesion inhibitor NIF. J. Cell Biol. 127:20812091.[Abstract]
- Zhang, L., and E.F. Plow. 1996. A discrete site modulates activation of I domains. J. Biol. Chem. 271:2995329957.[Abstract/Free Full Text]
- Zhang, L., and E.F. Plow. 1999. Amino acid sequences within the alpha subunit of integrin alpha M beta 2 (Mac-1) critical for specific recognition of C3bi. Biochemistry. 38:80648071.[CrossRef][Medline]
- Rieu, P., T. Sugimori, D.L. Griffith, and M.A. Arnaout. 1996. Solvent-accessible residues on the metal ion-dependent adhesion site face of integrin CR3 mediate its binding to the neutrophil inhibitory factor. J. Biol. Chem. 271:1585815861.[Abstract/Free Full Text]
- McGuire, S.L., and M.L. Bajt. 1995. Distinct ligand binding sites in the I domain of integrin alpha M beta 2 that differentially affect a divalent cation-dependent conformation. J. Biol. Chem. 270:2586625871.[Abstract/Free Full Text]
- Zhang, L., and E.F. Plow. 1997. Identification and reconstruction of the binding site within
Mß2 for a specific and high affinity ligand, NIF. J. Biol. Chem. 272:1755817564.[Abstract/Free Full Text]
- Li, R., P. Rieu, D.L. Griffith, D. Scott, and M.A. Arnaout. 1998. Two functional states of the CD11b A-domain: correlations with key features of two Mn2+-complexed crystal structures. J. Cell Biol. 143:15231534.[Abstract/Free Full Text]
- Ustinov, V.A., and E.F. Plow. 2002. Delineation of the key amino acids involved in neutrophil inhibitory factor binding to the I-domain supports a mosaic model for the capacity of integrin alphaMbeta 2 to recognize multiple ligands. J. Biol. Chem. 277:1876918776.[Abstract/Free Full Text]
- Yakubenko, V.P., D.A. Solovjov, L. Zhang, V.C. Yee, E.F. Plow, and T.P. Ugarova. 2001. Identification of the binding site for fibrinogen recognition peptide gamma 383-395 within the alpha MI-domain of integrin alpha Mbeta 2. J. Biol. Chem. 276:1399514003.[Abstract/Free Full Text]
- Ault, K.A., and T.A. Springer. 1981. Cross-reaction of a rat-anti-mouse phagocyte-specific monoclonal antibody (anti-Mac-1) with human monocytes and natural killer cells. J. Immunol. 120:359364.
- Diamond, M.S., and T.A. Springer. 1993. A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen. J. Cell Biol. 120:545556.[Abstract]
- Robinson, M.K., D. Andrew, H. Rosen, D. Brown, S. Ortlepp, P. Stephens, and E.C. Butcher. 1982. Antibody directed against the Leu-CAM b-chain (CD18) promotes both LFA-1- and CR3-dependent adhesion events. J. Immunol. 148:10801085.
- Dransfield, I., and N. Hogg. 1989. Regulated expression of Mg2+ binding epitope on leukocyte integrin alpha subunits. EMBO J. 8:37593765.[Abstract]
- Simon, D.I., N.K. Rao, H. Xu, Y. Wei, O. Majdic, E. Ronne, L. Kobzik, and H.A. Chapman. 1996. Mac-1 (CD11b/CD18) and the urokinase receptor (CD87) form a functional unit on monocytic cells. Blood. 88:31853194.[Abstract/Free Full Text]
- Zhang, L., and E.F. Plow. 1996. Overlapping, but not identical, sites are involved in the recognition of C3bi, neutrophil inhibitory factor, and adhesive ligands by the alphaMbeta2 integrin. J. Biol. Chem. 271:1821118216.[Abstract/Free Full Text]
- Lu, H., C.W. Smith, J. Perrard, D. Bullard, L. Tang, S.B. Shappell, M.L. Entman, A.L. Beaudet, and C.M. Ballantyne. 1997. LFA-1 is sufficient in mediating neutrophil emigration in Mac-1-deficient mice. J. Clin. Invest. 99:13401350.[Abstract/Free Full Text]
- Dallacqua, W., E.R. Goldman, E. Eisenstein, and R.A. Mariuzza. 1996. A mutational analysis of the binding of two different proteins to the same antibody. Biochemistry. 35:96679676.[CrossRef][Medline]
- Luscinskas, F.W., G.S. Kansas, H. Ding, P. Pizcueta, B.E. Schleiffenbaum, T.F. Tedder, and M.A. Gimbrone, Jr. 1994. Monocyte rolling, arrest and spreading on IL-4activated vascular endothelium under flow is mediated via sequential action of L-selectin, ß 1-integrins, and ß 2-integrins. J. Cell Biol. 125:14171427.[Abstract]
- Landis, R.C., A. McDowall, C.L. Holness, A.J. Littler, D.L. Simmons, and N. Hogg. 1994. Involvement of the "I" domain of LFA-1 in selective binding to ligands ICAM-1 and ICAM-3. J. Cell Biol. 126:529537.[Abstract]
- Wright, S.D., J.S. Weitz, A.J. Huang, S.M. Levin, S.C. Silverstein, and J.D. Loike. 1988. Complement receptor type three (CD11b/CD18) of human polymorphonuclear leukocytes recognizes fibrinogen. Proc. Natl. Acad. Sci. USA. 85:77347738.[Abstract]
- Altieri, D.C., R. Bader, P.M. Mannucci, and T.S. Edgington. 1988. Oligospecificity of the cellular adhesion receptor Mac-1 encompasses an inducible recognition specificity for fibrinogen. J. Cell Biol. 107:18931900.[Abstract]
- Diamond, M.S., D.E. Staunton, S.D. Marlin, and T.A. Springer. 1991. Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation. Cell. 65:961971.[Medline]
- Altieri, D.C., J.H. Morrissey, and T.S. Edgington. 1988. Adhesive receptor Mac-1 coordinates the activation of factor X on stimulated cells of monocytic and myeloid differentiation: an alternative initiation of the coagulation cascade. Proc. Natl. Acad. Sci. USA. 85:74267466.[Abstract]
- Wachtfogel, Y.T., R.A. DeLa Cadena, S.P. Kunapuli, L. Rick, M. Miller, R.L. Schultz, D.C. Altieri, T.S. Edgington, and R.W. Colman. 1994. High molecular weight kininogen binds to Mac-1 on neutrophils by its heavy chain (domain 3) and its light chain (domain 5). J. Biol. Chem. 269:1930719312.[Abstract/Free Full Text]
- Arnaout, M.A. 1990. Leukocyte adhesion molecules deficiency: its structural basis, pathophysiology and implications for modulating the inflammatory response. Immunol. Rev. 114:145180.[Medline]
- Plow, E.F., and L. Zhang. 1997. A MAC-1 attack: integrin functions directly challenged in knockout mice. J. Clin. Invest. 99:11451146.[Free Full Text]
- Coxon, A., P. Rieu, F.J. Barkalow, S. Askari, A.H. Sharpe, U.H. von Andrian, M.A. Arnaout, and T.N. Mayadas. 1996. A novel role for the beta 2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation. Immunity. 5:653666.[Medline]
- Shi, C., X. Zhang, Z. Chen, M.K. Robinson, and D.I. Simon. 2001. Leukocyte integrin Mac-1 recruits toll/interleukin-1 receptor superfamily signaling intermediates to modulate NF-kappaB activity. Circ. Res. 89:859865.[Abstract/Free Full Text]
- Santoso, S., U.J. Sachs, H. Kroll, M. Linder, A. Ruf, K.T. Preissner, and T. Chavakis. 2002. The junctional adhesion molecule 3 (JAM-3) on human platelets is a counterreceptor for the leukocyte integrin Mac-1. J. Exp. Med. 196:679691.[Abstract/Free Full Text]
- Huizinga, E.G., S. Tsuji, R.A. Romijn, M.E. Schiphorst, P.G. de Groot, J.J. Sixma, and P. Gros. 2002. Structures of glycoprotein Ibalpha and its complex with von Willebrand factor A1 domain. Science. 297:11761179.[Abstract/Free Full Text]
- Forsyth, C.B., E.F. Plow, and L. Zhang. 1998. Interaction of the fungal pathogen Candida albicans with integrin CD11b/CD18: recognition by the I domain is modulated by the lectin-like domain and the CD18 subunit. J. Immunol. 161:61986205.[Abstract/Free Full Text]
- Xie, J., R. Li, P. Kotovuori, C. Vermot-Desroches, J. Wijdenes, M.A. Arnaout, P. Nortamo, and C.G. Gahmberg. 1995. Intercellular adhesion molecule-2 (CD102) binds to the leukocyte integrin CD11b/CD18 through the A domain. J. Immunol. 155:36193628.[Abstract]
- Ostrovsky, L., A.J. King, S. Bond, D. Mitchell, D.E. Lorant, G.A. Zimmerman, R. Larsen, X.F. Niu, and P. Kubes. 1998. A juxtacrine mechanism for neutrophil adhesion on platelets involves platelet-activating factor and a selectin-dependent activation process. Blood. 91:30283036.[Abstract/Free Full Text]
- Diacovo, T.G., A.R. de Fougerolles, D.F. Bainton, and T.A. Springer. 1994. A functional integrin ligand on the surface of platelets: intercellular adhesion molecule-2. J. Clin. Invest. 94:12431251.[Medline]
- Rogers, C., E.R. Edelman, and D.I. Simon. 1998. A monoclonal antibody to the ß2-leukocyte integrin Mac-1 (CD11b/CD18) reduces intimal thickening after angioplasty or stent implantation in rabbits. Proc. Natl. Acad. Sci. USA. 95:1013410139.[Abstract/Free Full Text]
- Simon, D.I., Z. Chen, P. Seifert, E.R. Edelman, C.M. Ballantyne, and C. Rogers. 2000. Decreased neointimal formation in Mac-1(-/-) mice reveals a role for inflammation in vascular repair after angioplasty. J. Clin. Invest. 105:293300.[Abstract/Free Full Text]
- 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. 301:15311534.[Abstract/Free Full Text]