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Blood, 1 May 2007, Vol. 109, No. 9, pp. 3725-3732. Prepublished online as a Blood First Edition Paper on January 5, 2007; DOI 10.1182/blood-2006-11-058420. This Article Abstract Full Text (PDF) Supplemental Figures All Versions of this Article: blood-2006-11-058420v1 109/9/3725    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Beau Mitchell, W. Articles by Coller, B. S. Search for Related Content PubMed PubMed Citation Articles by Beau Mitchell, W. Articles by Coller, B. S. Related Collections Hemostasis, Thrombosis, and Vascular Biology Cell Adhesion and Motility Signal Transduction Gene Expression Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Mapping early conformational changes in IIb and ß3 during biogenesis reveals a potential mechanism for IIbß3 adopting its bent conformation W. Beau Mitchell1,2, Jihong Li2, Marta Murcia2,3, Nathalie Valentin4, Peter J. Newman5, and Barry S. Coller2 1 Department of Pediatrics, Mount Sinai School of Medicine, New York, NY; 2 Laboratory of Blood and Vascular Biology, Rockefeller University, New York, NY; 3 Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY; 4 Laboratoire d'Immunologie, Centre Hospitalier Universitaire, Nantes, France; 5 Blood Center of Wisconsin, Milwaukee, WI    Abstract Top Abstract Introduction Materials and methods Results Discussion Authorship References   Current evidence supports a model in which the low-affinity state of the platelet integrin IIbß3 results from IIbß3 adopting a bent conformation. To assess IIbß3 biogenesis and how IIbß3 initially adopts the bent conformation, we mapped the conformational states occupied by IIb and ß3 during biogenesis using conformation-specific monoclonal antibodies (mAbs). We found that IIbß3 complex formation was not limited by the availability of either free pro-IIb or free ß3, suggesting that other molecules, perhaps chaperones, control complex formation. Five ß3-specific, ligand-induced binding site (LIBS) mAbs reacted with much or all free ß3 but not with ß3 when in complex with mature IIb, suggesting that ß3 adopts its mature conformation only after complex formation. Conversely, 2 IIb-specific LIBS mAbs directed against the IIb Calf-2 region adjacent to the membrane reacted with only minor fractions of free pro-IIb, raising the possibility that pro-IIb adopts a bent conformation early in biogenesis. Our data suggest a working model in which pro-IIb adopts a bent conformation soon after synthesis, and then ß3 assumes its bent conformation by virtue of its interaction with the bent pro-IIb.    Introduction Top Abstract Introduction Materials and methods Results Discussion Authorship References   Integrin receptors are composed of heterodimers of and ß subunits. The ß3 family of integrin receptors is composed of IIbß3, which is specific for platelets and their precursor megakaryocytes, and vß3, which is more widely distributed on many cells, including osteoclasts and endothelial cells.1 IIbß3 plays an important role in platelet aggregation, and inhibitors of the receptor have demonstrated efficacy in preventing thrombotic complications of percutaneous coronary interventions.2 Previous studies of IIbß3 integrin biogenesis have provided important information on the synthesis of the IIb and ß3 subunits, IIbß3 complex formation, carbohydrate processing, transport of the assembled complexes from the endoplasmic reticulum (ER) to the Golgi, cleavage of IIb into heavy and light chains in the Golgi, and subsequent transport of the mature receptor to granule and platelet surface membranes.3–11 In particular, they suggested that the availability of either free pro-IIb or free ß3 limits IIbß3 complex formation.7,9 The crystal structure of the extracellular domain of vß3 unexpectedly revealed that the receptor had a bent conformation,12 and electron microscopy suggested that activation and ligand binding are associated with the adoption of an extended conformation.13 It is presumed that IIbß3 also undergoes a transformation from a bent to an extended conformation on activation,14 which occurs when platelets are stimulated with one or more agonists, resulting in binding of ligands, including fibrinogen. The crystal structure of the headpiece of IIbß3 in complex with ligand-mimetics also identified a swing-out motion of the ß3 hybrid domain at its juncture with the ßA (I-like) domain that is thought to contribute to ligand binding and/or the initiation of outside-in signaling.15 The bent, inactive conformations of these integrins thus play an important role in the function of these receptors. In the case of IIbß3, it permits platelets to circulate in plasma containing high concentrations of one of its ligands, fibrinogen, without spontaneous aggregation. Thus, it is important to understand how the bent, compact conformation is achieved during biogenesis (Figure 1). View larger version (46K): [in this window] [in a new window]   Figure 1. Progression of IIbß3 conformations during biogenesis. (A) The conformations of free pro-IIb, free ß3, and pro-IIbß3 are unknown. The conformation of mature, bent, unactivated IIbß3 and the proposed extended structure of activated, ligand-bound IIbß3 are modeled after the crystal structures of Vß3 and the IIbß3 headpiece, respectively. The domains of IIb and ß3 are indicated.12,15 (B) Immunoprecipitation with IIbß3 complex-specific mAbs 10E5 and 7E3 following pulse-chase labeling demonstrates that both antibodies recognize the pro-IIbß3 complex in addition to the mature IIbß3 complex. The figure on the left indicates the locations of the 10E5 and 7E3 epitopes on models of the extended and bent forms of IIbß3. This figure has been adapted from its original published form with the permission of the journal Clinical Pharmacology & Therapeutics.   We recently reported that the IIb N-linked glycan at amino acid 15, which is located on a solvent-exposed loop in the ß-propeller domain, is important for IIbß3 complex formation, presumably by mediating attachment to the ER membrane-bound lectin calnexin.16 In the present study we have used pulse-chase analysis of HEK293 cells synthesizing IIb and ß3, as well as megakaryocyte lineage cells expressing IIbß3 derived from umbilical cord blood and a series of monoclonal antibodies (mAbs) specific for different regions and conformations of IIb and ß3 to chart the progress of IIb and ß3 folding during biogenesis. Our data demonstrate that neither free pro-IIb nor free ß3 limits pro-IIbß3 complex formation, indicating that control of this vital step in biogenesis is achieved by some other mechanism, perhaps the availability of one or more chaperone molecules. In addition, the mAb immunoprecipitation patterns support a model in which pro-IIb becomes bent during or immediately after synthesis, perhaps as a result of the engagement of the head region, which is synthesized first, with the membrane-bound lectin/chaperone calnexin. The ß3 subunit achieves its final bent conformation only after complexing with pro-IIb.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion Authorship References   Antibodies The antibodies used in these studies and their specificities are detailed in Table 1 based on previous reports17–25 and the current studies. Several of the mAbs (AP5, AP6, LIBS1, LIBS2, PMI-1, B1B5) are categorized as ligand-induced binding site (LIBS) antibodies because they preferentially or exclusively bind to the activated and/or ligand-bound conformation(s) of IIbß3.18,21 View this table: [in this window] [in a new window]   Table 1. Specificities of antibodies used in this study   Human umbilical cord blood culture Leukocytes were separated from 3 to 6 units of human umbilical cord blood judged to be inadequate for clinical purposes (generously provided by the New York Blood Center) by Dextran 70 sedimentation (Amersham Biosciences, Piscataway, NJ), and then enriched for CD34+ progenitor cells by negative selection using a combination of antibodies against maturation/lineage-specific markers (RosetteSep; StemCell Technologies, Vancouver, BC) concomitant with density sedimentation (Ficoll-Paque Plus; Amersham Biosciences). These cells were then cultured in serum-free medium (StemCell Technologies) with 50 ng/mL thrombopoietin (TPO) plus 10 ng/mL IL-11 for 3 days (both StemCell Technologies), followed by culture in the same medium with 50 ng/mL TPO alone for another 6 to 8 days. HEK293-cell culture HEK293 cell lines that stably expressed normal human IIbß3 receptors were established as previously described.30 Transfections were performed using Lipofectamine 2000 (Gibco-BRL, Carlsbad, CA) according to the manufacturer's instructions, followed by selection in media containing 800 µg/mL G418 for 2 to 4 weeks. To obtain a population of cells uniformly expressing high levels of IIbß3, cells were labeled with the mAb 10E5 (anti-IIbß3) and sorted using a FACSVantage SE cell sorter (Becton-Dickinson, Rutherford, NJ). Biosynthetic labeling and immunoprecipitation Samples were prepared as previously described.34 Briefly, cells were incubated for 30 minutes at 37°C in methionine/cysteine-free medium, followed by pulse-labeling for 15 minutes at 37°C in medium containing 35S-methionine/cysteine (300 µCi [11.1 MBq]/10 cm plate). The pulse was terminated by incubation in medium containing unlabeled methionine/cysteine (1 mg/mL each), and the cells were incubated at 37°C until lysis in 1% Triton-X 100 lysis buffer. Following cell lysis, supernatants were precleared with protein-G Sepharose beads (Amersham Biosciences), and samples containing equivalent amounts of trichloroacetic acid-precipitable radioactivity ( 5-6 x 106 counts/sample) were incubated 16 hours at 4°C with one or more of the antibodies listed under "Antibodies" (4 µg/reaction). Samples were incubated with protein-G Sepharose beads for 1 hour at 4°C, washed twice, and incubated with SDS sample buffer for 10 minutes at 100°C. Samples were then subjected to SDS–polyacrylamide gel electrophoresis, and the gels were dried and exposed to film. To ensure that equal amounts of protein were loaded into each well, duplicate samples were analyzed by immunoblotting. The amount of each mAb used for immunoprecipitation was determined to be at a near-saturating concentration by titration experiments using 0 to 20 µg of each mAb (data not shown). Nonspecific binding was determined by performing immunoprecipitation with mouse IgG on whole-cell lysates of both cell types. Computer modeling of IIb amino acids A complete model of the structure of the extracellular inactive conformation of IIbß3 was first constructed using MODELLER 8v2.35 The IIb chain was modeled taking the coordinates of the propeller from the crystal structure of IIbß3 (residues 1-453; PDB ID, 1TY6)15 and using V coordinates as a template for the remainder of the sequence (PDB ID, 1U8C).36 Coordinates of the ß3 chain were extracted from the complex of Vß3 (PDB ID, 1U8C),36 and the missing domains, EGF1 (435-475) and EGF2 (486-522), were modeled from the crystal structures of the ß2 EGF1 (PDB ID, 1YUK)37 and the Vß3 EGF3 (PDB ID, 1U8C) domains,36 respectively. The complete system (including cations from the Vß3 crystal structure) was then energy minimized using GROMACS 3.2.138 as follows: (1) the system was immersed in a preequilibrated 16 x 16 x 16 nm cubic box of simple point-charge water molecules, and sodium (44) counter-ions were added to neutralize the system; (2) surrounding water molecules were first minimized and then subjected to 50 ps molecular dynamics simulations at 300°K and 1 bar, while harmonically restraining all protein heavy atoms to their initial positions [force constant equal to 1000 kJ/(mol · nm2)]; and (3) the resulting system was energy minimized without any restraints. To assess the most likely conformations of the region of V corresponding to amino acids 842 to 873 in IIb, a set of 200 independently optimized conformations of the 842 to 873 loop in the Calf-2 domain were generated from the minimized model of IIbß3 using the loop modeling routine of MODELLER 8v2.35 Briefly, the optimization relies on a protocol consisting of conjugate gradient minimization and molecular dynamics simulation with simulated annealing. The final loop prediction is the optimized conformation that has the lowest pseudo-energy score, which contains terms from a molecular mechanics force field as well as restraints based on statistical distributions derived from known protein structures. Because the accuracy of the MODELLER algorithm was only established for loops that are much shorter (up to 14 residues) than the one we modeled (32 residues), we also used the Robetta server39 to obtain 5 comparative models of the structure of the Calf-2 domain (residues 745 to 960) based on the corresponding V structure (PDB ID, 1JV2).39 Robetta assembles loop regions from fragments and optimizes them to fit the aligned template structure using the Rosetta fragment insertion method.40 This algorithm has been reported to provide reasonable models of conformations of peptide segments containing 13 to 34 residues.41 Multiple decoy models were generated and independent simulations were carried out. From this ensemble 4 models were selected (models 1-4) using different variants of the Rosetta energy function and are presented along with the default K*Sync alignment-derived model (model 5).42    Results Top Abstract Introduction Materials and methods Results Discussion Authorship References   Experiments with HEK293 cells transfected with IIb and ß3 IIbß3 Complex-dependent antibodies 10E5 and 7E3 react with pro-IIbß3 in addition to mature IIbß3. To assess complex formation between IIb and ß3 during biogenesis, we used 2 mAbs, 10E5 and 7E3, that react with the mature IIbß3 complex17,19 but not with either subunit alone.43 10E5 interacts exclusively with the "Cap" subdomain of the IIb ß-propeller,15 whereas the 7E3 epitope reacts with a region adjacent to the MIDAS in the ßA (I-like) domain of ß3.30 Their patterns of precipitation of radiolabeled IIb and ß3 were nearly identical (Figure 1). At the 1-hour time point they precipitated pro-IIb, mature IIb, and ß3; at 2 and 4 hours the intensity of the pro-IIb band progressively decreased, and the intensities of the mature IIb and ß3 bands increased, consistent with ongoing IIbß3 biogenesis during this time period.7,16 There was minimal further increase in IIb and ß3 band intensity after 4 hours (data not shown), indicating that biogenesis was essentially complete by this time point. Immunodepletion by 10E5 removed the IIb and ß3 species recognized by 7E3 and vice versa (data not shown). Thus, both of these antibodies recognize pro-IIbß3 complexes in addition to mature IIbß3 complexes. Anti-IIb antibodies CA3 and anti-V5 identify an excess of free pro-IIb. To assess the presence of a pool of pro-IIb that is not complexed with ß3, we used the anti-IIb–specific mAbs CA3, whose epitope is unknown, and anti-V5, which is directed at a V5 tag added to the cytoplasmic domain of the recombinant IIb used to transfect the cells. Each of these mAbs precipitated more pro-IIb than did either 10E5 or 7E3, but similar amounts of mature IIb and ß3, at both the 2- and 4-hour time points (compare Figure 1 with Figure 2B). Thus, they react with free pro-IIb, the pro-IIbß3 complex, and the mature IIbß3 complex. The amount of pro-IIb precipitated at 4 hours was less than that precipitated at 2 hours, consistent with both conversion of pro-IIb into mature IIb and/or degradation of pro-IIb. Thus, there is a substantial pool of free pro-IIb at the 4-hour time point, even though production of mature IIbß3 appears to be essentially complete by that time. Therefore, the availability of pro-IIb does not appear to limit IIbß3 biogenesis. View larger version (35K): [in this window] [in a new window]   Figure 2. Excess free pro-IIb and ß3 exist in stably transfected HEK293 cells. (A) Diagram localizing the epitopes of antibodies 10E5, 7E3, anti-V5, and 7H2 on the bent and the extended and ligand-bound conformations of IIbß3; the epitope of antibody CA3 is on IIb, but it has not been localized to a specific region. (B) Two anti-IIb antibodies, CA3 and anti-V5, immunoprecipitated more pro-IIb at both 2 hours and 4 hours than the complex-specific antibody 10E5. (C) Anti-ß3 antibody 7H2 bound to ß3 that was in complex with either pro-IIb or mature IIb and also immunoprecipitated more ß3 at both 2 hours and 4 hours than the 2 complex-specific antibodies 10E5 (Figure 2B) and 7E3. All gel lanes in this figure are from the same immunoblot. Equivalent amounts of protein were loaded in each lane.   Anti-ß3-specific antibodies, 7H2 and AP3, identify excess free ß3. To assess the presence of a pool of ß3 not complexed with IIb, we used the ß3-specific mAbs 7H2 and AP3. 7H2 appears to react with an epitope on or near the PSI domain, because a C13A mutation in this domain results in loss of 7H2 binding (W.B.M., J.L., and B.S.C., unpublished observations, April 2005). The mAb AP3 reacts with an epitope that includes amino acids 50 and 62 from the PSI and hybrid domains, respectively20,31,44 (J. Peterson and P.J.N., unpublished observations). 7H2 precipitated more ß3 than either 10E5 or 7E3 but similar amounts of pro-IIb and mature IIb (Figure 2C), and AP3 precipitated ß3 with virtually the same pattern. Thus, these mAbs react with free ß3 in addition to ß3 in complex with either pro-IIb or mature IIb. These data indicate that there also is a substantial pool of free ß3 at the 4-hour time point, even though production of mature IIbß3 appears to be essentially complete by this time. Thus, the availability of ß3 does not appear to limit IIbß3 biogenesis. Epitopes of the anti-ß3 LIBS-specific antibodies are exposed on both free ß3 and a subpopulation of the pro-IIbß3 complex. Antibodies AP5 and LIBS2 have previously been shown to recognize epitopes on the ß3 PSI and ßTD domains, respectively (Figure 3A). 22,32,33,44 They bind to only a small fraction of mature IIbß3 complexes on unactivated platelets, and their binding is markedly enhanced by ligand binding to the receptor or by EDTA treatment. After 2 to 4 hours of chase, both of these antibodies precipitated nearly as much ß3 as the non-LIBS mAb 7H2 (Figure 3B). They also precipitated some pro-IIb [but less than that precipitated by mAbs 10E5 (data not shown) and 7H2 (Figure 3B)] but no mature IIb. Thus, after considering the amount of ß3 that is precipitated by 7H2 as part of the mature IIbß3 complex, it appears that AP5 and LIBS2 react with virtually the entire population of free ß3 and subpopulations of the pro-IIbß3 complex. These data indicate that (1) the epitopes for these antibodies on the PSI and ßTD domains are exposed and accessible on free ß3; (2) on pro-IIbß3 complex formation, ß3 subunits undergo conformational changes that begin to mask or alter the AP5 and LIBS2 epitopes, and/or these epitopes are blocked by association with pro-IIb; and (3) the conformational changes leading to loss or masking of these epitopes are complete on formation of mature IIbß3. View larger version (47K): [in this window] [in a new window]   Figure 3. ß3-Specific LIBS antibodies variably recognize free ß3 and the pro-IIßß3 complex but not the mature IIbß3 complex. (A) Epitopes of mAb 7E3, 7H2, and the ß3-specific LIBS antibodies are indicated by spheres on the bent and extended structures of ligand-bound IIbß3. The epitopes of LIBS1 and PMI-2 are unknown. (B) Precipitation of IIb and ß3 by the mAbs at various times after initiating the chase. The mAbs LIBS2 and AP5 precipitated virtually the entire pool of free ß3 (defined by the binding of the non-LIBS mAb 7H2), a portion of the pro-IIb in complex with ß3 but no mature IIbß3. The mAbs PMI-2, LIBS1, and LIBS6 precipitated subpopulations of uncomplexed ß3 but no complexed ß3. Data shown are from one of more than 5 experiments. The top row of blots is from a single experiment and is overexposed to show the fainter pro-IIb bands. The bottom row is from a separate experiment and earlier time point to show the absence of mature IIb in the AP5 precipitate. Equivalent amounts of protein were loaded in each lane.   Anti-ß3 LIBS-specific antibodies PMI-2, LIBS1, and LIBS6 react with free ß3, but not with the pro-IIbß3 or mature IIbß3 complexes. The mAb LIBS6 reacts within the ßTD domain of ß3, near the transmembrane domain,22,25 whereas the epitopes of PMI-2 and LIBS1, both of which inhibit platelet aggregation, are not known.22,44,45 These mAbs bind to only a small percentage of unactivated mature IIbß3 receptors, and their binding is greatly enhanced by ligand binding to IIbß3.18,22,45 Each of these antibodies precipitated ß3 at 2 and 4 hours (Figure 3B). Unlike mAbs 7H2, 7E3, AP5, and LIBS2, however, these antibodies did not precipitate pro-IIb (even when longer exposures were analyzed to adjust for the lower density of the ß3 bands), and unlike mAbs 7H2 and 7E3 they did not precipitate mature IIb. Thus, the epitopes for these mAbs are exposed on both ligand-bound and free ß3, but not ß3 in complex with either pro-IIb or mature IIb. To estimate the fraction of free ß3 precipitated by these antibodies, the intensity of the ß3 band precipitated by each antibody was divided by the difference in intensity of the ß3 bands precipitated by 7H2 (representing free ß3 plus ß3 in complex with IIb) and 7E3 (representing ß3 in complex with IIb). These values were 65% for PMI-2, 75% for LIBS1, and 40% for LIBS6. Epitopes of 3 anti-IIb LIBS-specific antibodies are masked on free pro-IIb, pro-IIbß3 and mature IIbß3. Antibody PMI-1 has previously been shown to react with a linear sequence in IIb near the IIb cleavage site (amino acids 844-859).18,46 Although the homologous region in V was not defined in the V crystal structure,12,47 it is contained in a loop, and the amino acids on either side of the loop map to the base of the Calf-2 domain (Figure 4A). Binding of mAb PMI-1 to IIb is enhanced by ligand binding or EDTA treatment of IIbß3.18,21 B1B5 recognizes the IIb light chain between residues 859 and 993 in the Calf-2 domain29 (Figure 4A). The binding of B1B5 to platelets is enhanced by incubation with tirofiban (2-fold) or treatment with either EDTA (1.5-fold) or DTT (2-fold), thus identifying B1B5 as a partial LIBS mAb (W.B.M., J.L., and B.S.C., unpublished data, March 2006). Both PMI-1 and B1B5 give strong signals on immunoblots of IIb: PMI-1 reacts with the heavy chain and B1B5 reacts with the light chain (Figure S1, available on the Blood website; see the Supplemental Figures link at the top of the online article). View larger version (42K): [in this window] [in a new window]   Figure 4. IIb-Specific LIBS mAbs recognize only small amounts of free pro-IIb. (A) Epitopes of the mAb anti-V5 and the IIb-specific LIBS mAbs are indicated by spheres or bars on the bent, unactivated, and the proposed extended structure of ligand-bound IIbß3. (B) IIb-Specific LIBS mAbs B1B5 and PMI-1 reacted with subpopulations of free pro-IIb, pro-IIbß3, and mature IIbß3. Polyclonal antibody anti-IIb (H-160), whose epitope encompasses both the B1B5 and PMI-1 epitopes, precipitated less pro-IIb than V5. The 1.5-hour time point is depicted from 1 of more than 5 experiments. Equivalent amounts of protein were loaded in each lane. (C) PMI-1 precipitated less pro-IIb than CA3 from cells with an IIbN15Q mutation that eliminates the N15 glycan.   Both PMI-1 and B1B5 precipitated less pro-IIb than did mAbs anti-V5 or CA3 at all time points (Figure 4B and data not shown), with PMI-1 precipitating less pro-IIb than B1B5 in 6 of 6 experiments. PMI-1 also precipitated much less mature IIb and ß3 than did either anti-V5 or CA3, or the complex-dependent mAbs 10E5 and 7E3 (Figures 2B-C and 4B and data not shown). mAb B1B5 precipitated more pro-IIb and mature IIb than PMI-1, but less than the amount precipitated by anti-V5, consistent with its partial LIBS characteristics. The rabbit polyclonal antibody, anti-IIb (H-160), prepared against IIb amino acids 847 to 1006, reacted strongly with the IIb heavy chain (Mr 120 kDa) on immunoblot, indicating that it recognizes an epitope close to that of PMI-1, and only minimally with the IIb light chain (Mr 22 kDa) (data not shown). Anti-IIb (H-160) immunoprecipitated only a subpopulation of free pro-IIb and virtually none of the IIb in complex with ß3. When compared with the density of the pro-IIb band precipitated by anti-V5, the density of the bands precipitated by PMI-1, B1B5, and H-160 were 21% ± 18%, 45% ± 6%, and 54% ± 34% of the density of the V-5 precipitated band (all n = 3). Thus, the PMI-1 and B1B5 epitopes are only expressed or available on subpopulations of free pro-IIb. Because all of these antibodies react with denatured IIb on immunoblots (Figure S1), the reduced immunoprecipitation is presumably due to decreased epitope expression or availability. To assess whether the PMI-1 epitope of the recombinant IIb could be exposed by EDTA, as it is on platelets, PMI-1 binding was determined by flow cytometry before and after treatment with EDTA. Only a small amount of PMI-1 bound to untreated cells, but EDTA treatment increased PMI-1 binding more than 5-fold (Figure S1). To assess the effect of eliminating the N15 glycan on recognition of pro-IIb by the LIBS mAb PMI-1, we performed immunoprecipitation studies on HEK293 cells expressing the IIb N15Q mutation in combination with normal ß3 (Figure 4C). At 45 minutes after pulse-chase, PMI-1 precipitated less pro-IIb than CA3 from cells expressing N15QIIbß3, but a slightly greater proportion of total pro-IIb (as judged by CA3 binding) than in cells expressing normal IIbß3. Experiments with megakaryocyte-lineage cells derived from umbilical cord blood Kinetics of IIbß3 biogenesis are similar in HEK293 and megakaryocyte-lineage cells derived from umbilical cord blood. To verify that observations made in transfected HEK293 cells reflect IIbß3 biogenesis in a more physiologically relevant cell, we studied biogenesis of IIbß3 in megakaryocyte-lineage cells derived from human umbilical cord blood (CB) cells. After 10 days of culture in the presence of TPO, 95% ± 2% of CB cells expressed IIbß3, 83% ± 5% expressed GPIb, and 54% ± 10% expressed 2ß1 (mean ± SD, all n = 4). On incubation with 10 µM thrombin receptor-activating peptide, the percentage of CB cells recognized by PAC1, an activation-dependant, ligand-mimetic anti-IIbß3 mAb, increased from 3% ± 2% to 16% ± 1% (mean ± SD, n = 3). The kinetics of ß3 production and pro-IIb conversion into mature IIb were similar in CB and HEK293 cells (compare Figures 1B and 5A). Pro-IIb disappearance, which reflects both the conversion of pro-IIb into mature IIb plus pro-IIb degradation, was slightly more rapid in CB cells [1.4 ± 0.2 hours (n = 4) versus 2.3 ± 1.0 hour (n = 3) for CB cells and stably transfected HEK293 cells, respectively, P = .1]. The immunoprecipitation patterns obtained with the mAb panel were similar between the CB and HEK293 cells. View larger version (51K): [in this window] [in a new window]   Figure 5. Kinetics of IIbß3 biogenesis in cells derived from umbilical cord blood (CB) cells. (A) Pulse-chase analysis of CB cells with the IIbß3 complex-specific mAb 10E5 showed a pattern of pro-IIb, mature IIb, and ß3 kinetics similar to those in stably transfected HEK293 cells (compare with Figure 2). Similar to the results in stably transfected HEK293 cells, the anti-IIb mAb CA3 and the anti-ß3 mAb 7H2 revealed the presence of pools of free pro-IIb and ß3, respectively, in the CB cells at a time when IIbß3 production was essentially complete. (B) The anti–ß3 LIBS mAbs AP5 and LIBS2 recognized pro-IIbß3 but not mature IIbß3, and the anti–IIb LIBS mAb PMI-1 precipitated only a small subpopulation of pro-IIb. Each box represents a separate experiment.      Discussion Top Abstract Introduction Materials and methods Results Discussion Authorship References   In this study, we used conformation-specific mAbs to assess the conformational changes that the pro-IIb and ß3 subunits undergo prior to and during formation of the pro-IIbß3 heterodimer and the mature IIbß3 receptor in a transfected cell line and in megakaryocyte-lineage cells derived from human umbilical cord blood. Previous studies of IIbß3 biogenesis4,7,9,10 established that (1) the 2 subunits are translated from separate mRNAs; (2) IIb has N-linked glycans that undergo processing in the Golgi, where IIb is cleaved into heavy and light chains; (3) ß3 is N-glycosylated but does not undergo processing of its N-linked glycans during receptor maturation; and (4) both IIb and ß3 are required for surface expression. Although the results of these studies were generally concordant, Duperray et al7 suggested that the rate of processing of IIb in chronic myelogenous leukemia–derived megakaryocytes may limit the production of IIbß3, whereas Rosa and McEver9 reported that in HEL cells the ß3 pool size may be limiting. We recently reported evidence that the pro-IIb N15 glycan on the IIb headpiece ß-propeller domain is important in IIbß3 biogenesis and proposed that interaction of the ß-propeller region with the membrane-associated chaperone calnexin may account for IIb adopting the bent, inactive conformation during biogenesis. In the current study, we identified sizable pools of both free pro-IIb and ß3 in 2 different cell types. Thus, pro-IIbß3 heterodimer formation does not appear to be limited by the availability of either subunit. Our data indicate that pro-IIbß3 complex formation is not controlled or limited by IIb subunit availability. Thus, factors other than subunit availability must control pro-IIbß3 complex formation; the availability of other chaperone molecules that mediate the binding of the ßA (I-like) domain of ß3 to the ß-propeller domain of IIb may be one of those factors. Our findings that mAbs 10E5 and 7E3 react with the pro-IIbß3 complex in addition to mature IIbß3,17,19 but not with free pro-IIb or free ß3, are similar to those reported for Lß2 by Huang et al, who used mAbs specific for the Lß2 complex with epitopes that are analogous to those of 10E5 and 78E3.48,49 They interpreted their data as indicating that the ßA (I-like) domain of ß2 and the ß-propeller domain of L do not become fully folded until complex formation occurs. This could also be the explanation for our findings. However, an alternative explanation for both their and our data is that the epitopes on the ß-propeller and ßA (I-like) domains are inaccessible to the mAbs because a chaperone(s) bind to the subunits in these regions. If this hypothesis is correct, then formation of the pro-IIbß3 complex most likely results from, or initiates, a conformational change that releases the complex from the chaperones, and this event may be a signal for transport to the Golgi for further processing. Data from a variety of techniques, including electron microscopy and x-ray crystallography, have led to a working model in which IIbß3 undergoes dramatic conformational changes with activation and ligand binding that involve leg separation, headpiece extension, and a ß3 swing-out motion at the junction of the ß3 ßA (I-like) domain and the hybrid domain.14,15 We have found that ß3 LIBS mAbs also bind most or all free ß3, and some also bind a portion of the pro-IIbß3 complex. Thus, it seems most likely that these LIBS epitopes become inaccessible after complex formation as a result of head-head, leg-leg, and/or head-leg interactions that stabilize the bent conformation of IIbß3 during the latter stages of biogenesis. Because mAbs AP5 and LIBS2 recognized virtually all of the free ß3 pool, their epitopes appear to be altered or masked exclusively by their association with IIß. In contrast, because LIBS1, LIBS6, and PMI-2 did not recognize all of the free ß3 pool, it is possible that either some ß3 leg-head interactions occur relatively early, or that chaperones or other adjacent molecules partially block access to these epitopes. Moreover, because the ß3 LIBS mAbs demonstrated variable reactivity with the pro-IIbß3 complex, it is possible that the ß3 regions recognized by the mAbs that are able to precipitate at least a fraction of the pro-IIbß3 complexes (PSI for AP5, and ßTD for LIBS2) are the last regions to adopt their mature conformations. Alternatively, separate subpopulations of pro-IIbß3 complexes may adopt different conformations after complex formation, either exposing or masking these epitopes. Of note, studies by Huang et al49 on Lß2 (LFA-1) biogenesis demonstrated that an activating mAb whose epitope localized to the hybrid/PSI/EGF region of ß2, recognized free ß2, but not ß2 in complex with L. These data are consistent with our results with mAb AP5, which is also an activating mAb and binds to the analogous region of ß3, and suggest that our observations on the step-wise nature of ß3 subunit association with IIb during biogenesis may be applicable to other integrin receptors. In contrast to anti-ß3 LIBS, the anti-IIb LIBS mAb PMI-1, whose epitope is near the IIb cleavage site within the Calf-2 domain,46 recognized approximately 20% of pro-IIb subunits. This suggests that the majority of pro-IIb subunits adopt a conformation that conceals the PMI-1 epitope immediately after subunit synthesis; that a chaperone, another molecule, and/or the platelet membrane conceals the epitope; or that the PMI-1 epitope is not formed until ligand binds to IIbß3. Because PMI-1 readily identifies both pro-IIb and mature IIb after denaturation in SDS by immunoblotting, and PMI-1 binds to a linear peptide,46 it seems more likely that its epitope is present but inaccessible. If the pro-IIb headpiece does bind to calnexin soon after synthesis, then the proximity between the membrane-associated headpiece and the tail region of IIb, which contains the PMI-1 epitope, may be responsible for the inaccessibility of the PMI-1 epitope. We also studied the binding of the antibody B1B5, whose epitope is on the extracellular portion of the IIb light chain, and the polyclonal anti-IIb antibody H-160, prepared against a peptide overlapping both the PMI-1 and B1B5 epitopes (Table 1). Like PMI-1, both of these antibodies precipitated less free pro-IIb than anti-V5. Although care must be used in the interpretation of decreased antibody binding, the finding that all 3 antibodies have limited reactivity is highly suggestive that the tail region of pro-IIb, which contains these epitopes, is not completely accessible during biogenesis. Because the Vß3 region corresponding to the IIb loop containing the PMI-1 epitope (844-859) was not identified in the Vß3 crystal structure,12 we do not have Vß3 structural information to help understand the conformation of the analogous region in IIb. To address this deficiency, we have applied 2 loop-determination programs, MODELLER 8v250 and Robetta,39 to predict the structure of the IIb loop between amino acids 842 to 873 in the Calf-2 domain, which encompasses the PMI-1 epitope, the dominant H-160 epitope(s), and perhaps some or all of the B1B5 epitope. It should be noted that reliable prediction of the conformation of a 32–amino acid loop is not possible at the present time because the accessible conformational space increases exponentially with increasing chain length. These data are presented only to suggest the likely conformational space available for this loop. Figure 6A shows the IIb subunit containing the conformation of the loop between amino acids 842 and 873 initially predicted by MODELLER, and Figure 6B shows the superimposition of 200 conformations for the loop, with the most favored shown in red, the least favored in blue, and those in between in green. Figure 5C shows 5 energetically favorable loop conformations derived using Robetta, a program that has been shown to provide reasonable structures for loops of 13 to 34 residues.41 The conformational space sampled by the Robetta loop modeling routine is quite broad and similar to that sampled by MODELLER. Some of the conformations from both programs that orient the loop below the Calf-2 region may not be possible in IIb on the platelet surface because of the location of the plasma membrane. Conformations that orient the loop between the ß-propeller and the Calf-2 domain, such as the red loop in Figure 6C, may not be accessible when the receptor is bent but may become accessible if the receptor adopts an extended position. In fact, Calzada et al51 reported that mAbs directed at epitopes on the ß-propeller domain could interfere with the binding of mAbs directed at epitopes on the Calf-2 domain, indicating the proximity of these regions in mature IIbß3 on the platelet surface. View larger version (62K): [in this window] [in a new window]   Figure 6. Computer models constructed using MODELLER or Robetta of the energetically most-favored conformations of the IIb loop between amino acids 842 and 873. (A) IIb with the loop (arrow) in an energetically favored conformation derived from MODELLER. The loop colors indicate an IIb cleavage site (R859) that separates the heavy (orange) and light (gray) chains. The PMI-1 epitope is immediately proximal to the cleavage site, as is at least a portion of the anti-IIb (H-160) epitope. The B1B5 epitope is on the light chain, indicated by the gray and green ribbons. (B) The 200 most energetically favorable conformations of the loop were derived using MODELLER. The most favorable conformations are in red, less favorable ones in green, and least favorable ones in blue. (C) Five loop conformations were derived using Robetta.   To obtain more information on the conformations of pro-IIb and mature IIb, we also assessed the ability of thrombin to cleave pro-IIb and mature IIb. Previous studies by Fujimura and Phillips52 and Phillips et al53 demonstrated that thrombin does not cleave mature IIb when it is complexed with ß3, but it does cleave mature IIb when the complex is dissociated with EDTA.52,53 The predicted thrombin cleavage sites in IIb are both within solvent-exposed regions of the ß-propeller that are not likely to be affected by the bending of IIb (Figure S2). However, they are likely to be masked by IIbß3 complex formation. We found that the free pro-IIb subunit is susceptible to thrombin cleavage, confirming that the cleavage sites are accessible in the free pro-IIb subunit (Figure S2). Thus, the inaccessibility of the PMI-1 and H-160 epitopes on the Calf-2 domain of free pro-IIb is specific to that region. If adoption of the bent conformation of IIb limits access to the Calf-2 domain, then our data suggest a working model in which pro-IIb adopts a bent conformation soon after synthesis, and then free ß3 assumes its bent conformation by virtue of its interaction with the bent pro-IIb. One possible mechanism by which pro-IIb could adopt a bent conformation would be through interaction of the pro-IIb headpiece with a membrane-associated chaperone, and we have previously proposed that the N15 glycan of IIb, which is important in IIbß3 biogenesis, interacts with the membrane-associated calnexin. In the current study, however, we did not observe a major increase in the fraction of pro-IIb precipitated by PMI-1 when the N15 glycan was eliminated by an N15Q substitution. Thus, it is possible that either the IIb headpiece can interact with some other membrane-associated chaperone, that pro-IIb interacts with calnexin via nonglycan interactions,54 or that pro-IIb headpiece association with the membrane is not necessary for limiting access to the pro-IIb Calf-2 domain. Additional studies are required to assess this working model. Understanding the biogenesis of integrins should provide insights into the mechanism(s) by which mutant receptors fail to form complexes or form abnormal complexes that do not progress to the Golgi. Such understanding may also provide insights into the energetics involved in the conformational changes associated with activation and ligand binding. Moreover, because antagonists to IIbß3, 4, and L have all demonstrated therapeutic efficacy,55–58 understanding integrin biogenesis may help in designing pharmacologic approaches to prevent integrin biogenesis or the conformational changes associated with integrin activation and/or ligand binding.    Authorship Top Abstract Introduction Materials and methods Results Discussion Authorship References   Contribution: W.B.M. designed and performed experiments, analyzed and interpreted data, and drafted the manuscript; J.L. and N.V. designed and performed experiments; M.M. performed molecular modeling; P.J.N. designed experiments and analyzed and interpreted data; B.S.C. designed experiments, analyzed and interpreted data, and drafted the manuscript. Conflict-of-interest disclosure: B.S.C. is an inventor of abciximab, a derivative of 7E3, and in accord with federal law and the policies of the Research Foundation of the State University of New York, shares in royalty payments made to the Foundation based on sales of abciximab. All other authors declare no competing financial interests. Correspondence: W. Beau Mitchell, New York Blood Center, 310 E 67th St, New York, NY 10021; e-mail bmitchell{at}nybloodcenter.org.    Acknowledgments   We thank Mortimer Poncz and Mark Ginsberg for providing antibodies, and the National Cord Blood Program of the New York Blood Center for providing umbilical cord blood. This work was supported in part by the National Institutes of Health (grants HL19278, CTSA-UL1RR024143, and GCRC-M01RR00102) (B.S.C.), (grant HL68622 01) (W.B.M.), and (grant HL44612) (P.J.N.) and by funds from Stony Brook University.    Footnotes   Submitted November 17, 2006; accepted December 20, 2006. 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