Molecular Dynamics Analysis of a Novel β3 Pro189Ser Mutation in a Patient with Glanzmann Thrombasthenia Differentially Affecting αIIbβ3 and αvβ3 Expression

Mutations in ITGA2B and ITGB3 cause Glanzmann thrombasthenia, an inherited bleeding disorder in which platelets fail to aggregate when stimulated. Whereas an absence of expression or qualitative defects of αIIbβ3 mainly affect platelets and megakaryocytes, αvβ3 has a widespread tissue distribution. Little is known of how amino acid substitutions of β3 comparatively affect the expression and structure of both integrins. We now report computer modelling including molecular dynamics simulations of extracellular head domains of αIIbβ3 and αvβ3 to determine the role of a novel β3 Pro189Ser (P163S in the mature protein) substitution that abrogates αIIbβ3 expression in platelets while allowing synthesis of αvβ3. Transfection of wild-type and mutated integrins in CHO cells confirmed that only αvβ3 surface expression was maintained. Modeling initially confirmed that replacement of αIIb by αv in the dimer results in a significant decrease in surface contacts at the subunit interface. For αIIbβ3, the presence of β3S163 specifically displaces an α-helix starting at position 259 and interacting with β3R261 while there is a moderate 11% increase in intra-subunit H-bonds and a very weak decrease in the global H-bond network. In contrast, for αvβ3, S163 has different effects with β3R261 coming deeper into the propeller with a 43% increase in intra-subunit H-bonds but with little effect on the global H-bond network. Compared to the WT integrins, the P163S mutation induces a small increase in the inter-subunit fluctuations for αIIbβ3 but a more rigid structure for αvβ3. Overall, this mutation stabilizes αvβ3 despite preventing αIIbβ3 expression.


Introduction
Glanzmann thrombasthenia (GT) is a rare inherited disease of platelet aggregation caused by quantitative and/or qualitative deficiencies of the aIIbb3 integrin [1][2][3]. The result is lifelong bleeding due to the inability of platelets to plug injured blood vessels. The ITGA2B and ITGB3 genes that encode aIIbb3 colocalize at chromosome 17q21.32 although their transcription is not coordinated [4]. Biosynthesis of aIIbb3 occurs in megakaryocytes (MKs) in the bone marrow; anucleate platelets are released in large numbers from protrusions called proplatelets extruded into the blood circulation [5]. GT is given by a large variety of nonsense and missense mutations, gene rearrangements including small insertions or deletions, splice site defects and frameshifts that occur across the 45 exons that compose ITGA2B and ITGB3 [2,3]. Whereas aIIb is mostly confined to the MK lineage, b3 is also present as avb3, a major integrin of vascular, blood and tissue cells; in contrast, avb3 is a very minor component in platelets [6][7][8]. Mutations in ITGA2B are specific for aIIbb3, but those effecting ITGB3 extend to both b3-containing integrins and potentially concern all cell types expressing avb3. While a majority of ITGB3 mutations affect b3 expression, missense mutations can have different effects on the capacity of b3 to interact with aIIb and av. Indeed, rare b3 mutations have been shown to allow avb3 expression while preventing the formation and/or maturation of aIIbb3. Alternatively, while permitting the expression of both integrins they may affect their function differently [9][10][11][12][13].
Elucidation of the crystal structures of the avb3 and aIIbb3 extracellular domains has allowed a close investigation of the interactions at the head domain interface between b3 and av or aIIb and has revealed distinct structural differences [14][15][16][17][18][19]. We now report studies that include a molecular dynamics analysis to investigate the effects on integrin structure of a novel b3Pro189Ser (P163S in the mature protein) mutation that we have located in a case of type I GT. This mutation prevents expression of the aIIbb3 complex while stabilizing the interaction between b3 and av.

Ethics Statement
Written informed consent was obtained from the patient prior to providing blood for the mutation analysis that was performed as part of the diagnosis of her disease. The patient herself reviewed her case report in the days preceding submittal of the manuscript. The study protocol was approved by the Human Research Ethics Committee of Alsace under the promotion of the French National Institute of Health and Medical Research (INSERM, Paris) under protocol RBM 04-14 for the French National Network for Disorders of Platelet Production and Function (Directors: JP Cazenave and AT Nurden) and was performed according to the Declaration of Helsinki.

Subjects
The propositus is a 49 year-old French woman of consanguineous parents who was diagnosed with GT when 5 years old (Case History S1). In brief, her platelets failed to aggregate with all physiologic agonists and failed to retract a clot. They minimally bound monoclonal antibodies (MoAbs) to aIIbb3 in flow cytometry despite a normal presence of other membrane glycoproteins ( Figure S1). aIIb was absent in western blotting performed using a polyclonal antibody to aIIbb3 with bound immunoglobulin located using 125 I-labeled Protein A as described [20]; however, residual b3 was present in low amounts and was of normal migration ( Figure 1A). As a further control for the specificity of antibody binding, we also studied in parallel platelets of a patient with a large ITGB3 deletion preventing b3 synthesis [21]. The residual b3 seen for the propositus suggested that avb3 was maintained, a finding confirmed for platelets by immunogoldlabelling and electron microscopy performed according to our standard procedures [8]. It should be noted that avb3 is organized essentially in intracellular vesicles as first described by us both in normal platelets and in another type I GT patient [8].

DNA analysis
Genomic DNA was extracted from 200 ml buffy coat (leukocyterich zone at the interface between platelet-rich plasma and red blood cells) of a centrifuged EDTA-anticoagulated blood sample, with a QiaAmpHDNA minikit (Qiagen S.A., Courtaboeuf, France) according to the manufacturer's protocol. Direct sequencing of all exons and splice sites of ITGA2B (30 exons) and ITGB3 (15 exons) was performed by the French National Sequencing Center (Génoscope, Evry, France). Briefly, exons and flanking regions of ITGA2B and ITGB3 were amplified by polymerase chain reaction (PCR) with a high fidelity Taq polymerase permitting large fragment amplification (TaKaRa LA TaqH DNA Polymerase, Millipore SA, Molsheim, France). PCR fragments were sequenced using the BigDye Terminator v3.1 Cycle reaction kit (Life Technologies, Saint Aubin, France) and a 3730 DNA Analyzer from Life Technologies. Further details of the Methods including the structure of all oligonucleotides are available on request. Pathogenicity of mutations was analyzed using Alamut Mutation Interpretation Software (Seine Biopolis, Rouen, France).
Chinese hamster ovary (CHO) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640-GlutaMAX TM (Gibco-Life Technologies, Paisley, UK) medium supplemented with 10% fetal calf serum. Cells were transiently transfected with empty plasmid (pcDNA3.1) or the WT-b3 or P163Sb3 expression plasmid either alone, or along with a WT-aIIb expression plasmid, pcDNA3.1-WTaIIb. For each well of a 6 well plate, a total of 2 mg of plasmid DNA was diluted in 500 ml of serum-free medium, before adding 5 ml of Lipofectamine LTX (Invitrogen-Life Technologies, Paisley, UK) and incubating the plate at room temperature for 25 minutes to allow formation of Lipofectamine-DNA complexes. CHO cells, grown to 80-90% confluence, were passaged and 1x10 5 cells, in 1.5 ml of complete medium, were added to each well, before incubating the plate at 37uC in the presence of 5% CO 2 . Forty eight hours after transfection, cells were harvested and expression of cell surface aIIbb3 assessed by flow cytometry on a FACSCalibur flow cytometer (BD Biosciences, Oxford, UK) using fluorescein isothiocyanate (FITC) conjugated anti-CD41 (MCA467F; AbD Serotec, Kidlington, UK) and phycoerythrin (PE) conjugated anti-CD61 (BD555754; BD Biosciences) monoclonal antibodies. Intracellular b3 expression was assessed similarly after fixing and rendering the cells permeable using the BD Cytofix/Cytoperm Kit (BD Biosciences). The ability of WT and b3P163S subunits to bind to av, expressed endogenously by CHO cells, was assessed using FITC conjugated monoclonal anti-avb3 (LM609; Chemicon, Chandlers Ford, UK).

Static Modeling of aIIbb3 and avb3
Models were obtained using the PyMol Molecular Graphics System, version 1.3, Schrödinger, LLC (www.pymol.org) and 3fcs and 1u8c pdb files for crystal structures of aIIb and av in complex with b3 in the bent conformation. Amino acids are visualized in the rotamer form showing side change orientations incorporated from the Dunbrack Backbone library with the maximum probability [2,3].

Molecular Dynamics Simulations
For aIIbb3 we started from the X-ray structure with PDB code 3NIG (resolution 2.25 Å ) and for avb3 with PDB code 3IJE (resolution 2.90 Å ). As b3 is very large and would have necessitated long simulation times we reduced the size of the structure examined. The GT database (http://sinaicentral.mssm. edu/intranet/research/glanzmann) shows that while a large majority of missense mutations are located in the ''head-groups'' of the two subunits, few are found at the N-terminal end of b3. Moreover, the distal extracellular b3-domain linked to the transmembrane sequence is free and being close to the a-subunit is prone to stick onto its surface during simulations leading to an abnormal complex. It was therefore decided to truncate b3 at residues 110 and 354. As a result, the extracellular N-terminal domain composing amino acids (aa) 1 to 110 and the membrane proximal C-terminal part represented by residues 354 to 466 were removed. The truncated b3 from P111 to S353 was used for aIIbb3 and avb3.
In order to check that the truncation of b3 had no detrimental influence on the behavior of the complexes, the two wild-type (WT) assemblies were submitted to a long (60 ns) molecular dynamics simulation. For this, the protein complex was centered in a rectangular water box with dimensions: 11061006100 Å . Then the whole system was neutralized and 150 mM NaCl added. This resulted in a box with 29,100 to 29,200 water molecules and approximately 184 NaCl molecules (the number may vary in the presence of the mutation). Calculations were accomplished using GROMACS 4.5 and the GROMOS96 force field (G43a1) packages [22]. The model for water was SPC (simple point charge). Molecular dynamics runs were performed at constant temperature (300 K, time constant for coupling tp = 0.1 ps) and pressure (P = 1 bar, tp = 0.5 ps) with a Berendsen coupling algorithm [23]. The time step = 2 fs, particle meshed Ewald (PME) method [24] was used with a cubic grid (1 Å ), Van der Waals (VDW) cut off = 10 Å , and frames were saved every 1000 steps.
In the same way, the b3 mutant P163S was created via the appropriate module in Discovery Studio version 3.1 and the two mutated complexes were submitted to identical molecular dynamics runs. One simulation was performed on truncated b3 alone, either WT or with the P163S mutation. Here we used a smaller water box with dimensions: 80680680 Å containing around 15,700 water molecules and 93 NaCl molecules. The molecular dynamics simulations were identical to the large box. In trajectory analyses root mean square deviations (RMSD) and root mean square fluctuations (RMSF) were calculated on Calpha positions as described in Jallu et al [25].

Molecular Characterization and Mutation Analysis
As shown in Figure 1A and Figure S1, platelets of the patient have a severe deficit in aIIbb3 that is characteristic of type I GT [1]. Small amounts of residual b3 were observed by Western blotting and avb3 was normally localized in her platelets by immunoelectron microscopy ( Figure 1B). This presence would suggest a genetic defect of ITGA2B. But unexpectedly, this was not confirmed by direct sequencing of ITGA2B (30 exons) and ITGB3 (15 exons) and their splice sites with results showing a homozygous C to T transition at position 565 of the cDNA (c.565C.T) within exon 4 of the ITGB3 gene. This gave a p.Pro189Ser substitution (P163S in the mature protein) ( Figure 1C). No other potential pathological mutations were located in either gene. Of interest, genotyping for the HPA1a/1b alloantigen system (L33P in the mature protein) carried by b3 showed homozygosity for the rare b3 HPA-1b alloantigen, a finding restricted to about 2% of Caucasians [26].
b3P163 is highly conserved within mammals and vertebrates ( Figure S2) and within different human b-subunits suggesting that it is important for integrin biosynthesis and/or function. According to the Alamut software, the physical and chemical deviation between a proline and a serine is important (Grantham score: 74) and according to the SIFT (sorting inherent from tolerant) score this mutation is predicted to be deleterious (SIFT score: 0.0).

Expression Studies in CHO Cells
The potential pathogenicity of the P163S substitution in b3 was further investigated after introduction of the mutation into a b3 Figure 1. Initial studies characterizing the molecular defect of aIIbb3 of the patient's platelets. A/Western blotting of aIIb and b3 in samples of SDS-soluble extracts of (a) control platelets (5mg protein), (b) the patient under study (60 mg) and (c) a second type I GT patient [21] with a large ITGB3 deletion preventing synthesis of b3 (60 mg). The integrin subunits were detected with a polyclonal antibody to aIIbb3 with bound IgG located using 125 I-labeled protein A. B/Immunogold labeling was performed on frozen-thin platelet sections using a pool of murine monoclonal antibodies specific for the av subunit [8] and bound antibody located for platelets from (a) a control donor and (b) from the patient using a speciesspecific second antibody to mouse IgG adsorbed on 5 nm gold particles and electron microscopy. Arrows highlight the largely vesicular distribution of avb3. C/Direct sequencing of genomic DNA of the patient (forward and reverse strands) for exon 4 of ITGB3. The nucleotide concerned by the mutation is framed. The patient is homozygous for a c.565 C/T transition leading to a p.Pro189Ser substitution (P163S in the primary b3 structure nomenclature). doi:10.1371/journal.pone.0078683.g001 expression construct and transient expression in CHO cells, either alone to give rise to a chimeric complex with endogenous hamster av, or with co-expression of normal human aIIb ( Figure 2). There was a 94% reduction in surface expression of the aIIbb3 receptor in CHO cells expressing P163Sb3 compared to those expressing WTb3 mirroring the deficit in expression of aIIbb3 observed in platelets from the patient with the defect (Figure 2A). In contrast, assessment of b3 after permeabilisation of the cells revealed that the b3P163S subunit was expressed intracellularly at 71% of the levels of those of WTb3 ( Figure 2B) and staining of avb3 on CHO cells transfected with b3 alone indicated that P163Sb3 was able to form a chimeric complex with hamster av which was expressed at 78% of the levels of the chimeric complex of WTb3 and hamster av ( Figure 2C). Moreover, labeling of cells transfected with the WT and P163Sb3 subunits alone, using the monoclonal antibody to b3 to detect the chimeric complex of avb3, showed no difference between cells expressing WTb3 and P163Sb3 subunits ( Figure 2D).

Static Modeling Analysis
Crystallography showed that b3P163 residue is situated at the interface between the aIIb or av and b3 subunit head domains [14,17]. This is illustrated by static modeling showing the contacts (colored) between the wild-type (WT) headpieces of aIIb and av (in blue) with b3 (in red) ( Figure 3A and B). Strikingly, the adjoining b3S162 is also mutated in a case of GT underlining the importance of this sequence situated in the b-I domain (see Discussion). A greater surface area and an increased number of amino acids participate in the interaction between b3 and aIIb compared to av both for the domain containing the b3P163S mutation (A.1 and B.1 windows) and in the entire complex between the headpieces. Significantly, as well as b3P163, amino acids engaged in H-bonds within aIIbb3 and avb3 are highly conserved through species ( Figure S2) although to a lesser extent within different a-subunits of the integrin family in man. Interestingly, this initial analysis highlighted only a single H-bond between b3P163 and H113 in av (see Box B1).

Molecular Dynamics Analysis
We then used molecular dynamics simulations to examine the effects of the P163S substitution on aIIbb3 and avb3 structure. We first plotted the RMSD (root mean square deviation) for each C-a position of wild type (WT) or P163S substituted b3 in complex with aIIb or av (Figure 4). Both integrin complexes show equivalent movements of the b3 backbone. The introduction of S163 induced only small changes in fluctuations at the site of the mutation (red arrows) for b3 in complex with aIIb or with av. However, more substantial changes occur approximately 100 amino acids onward with a dramatic increase in movements when mutated b3 is in complex with aIIb and, in contrast, a decrease and stabilization of the backbone structure when mutated b3 is associated with av (dotted box).
The influence of the P163S mutation on the secondary structures of b3 in complex with aIIb or av was then examined in timeline plots ( Figure 5). From left to right of these plots it is possible to follow the influence of the mutation on the secondary structure from the beginning (on the left) to the end of the dynamics run (after 50 ns of full dynamics). The main secondary structures (alpha helices in magenta and beta-strands in yellow) are largely unaffected by the mutation. Again, while the b3 secondary structure at the site of the substitution appears unaffected (red arrows), changes occur around 100 amino acids onwards (blue dotted box). Significantly, while a 3-10 amino acid a-helix (in blue) beginning at position 259 and framed by two b-turns (in green) can be clearly distinguished in WT aIIbb3, the presence of S163 results in a loss of the last b-turn. This latter structure is also lost when b3 is associated with av. Other differences are the loss of a small a-helix around position 229 for the a-subunit in avb3 and the appearance of a small a-helix around position 170 for the bsubunit in avb3. A 3-10 amino acid a-helix visible between position W129 and N148 was transient in nature when mutated b3 was in complex with aIIb and was lost after 10 to 15 ns of molecular dynamics, its significance is unknown.
In the WT integrins, the nature of the a-subunit clearly has a significant influence on the number of hydrogen bonds engaged by b3 (Table I) and confirms the static analysis. As measured in the last 6 ns of the molecular dynamics runs, in comparison to aIIbb3, avb3 shows a small global increase in H-bonds (+2.5%) and at the same time a marked decrease in the number of inter-subunit Hbonds (223.3%). This suggests either a decreased stability of WT avb3 compared to aIIbb3 or a weaker binding between the subunits in avb3. With b3S163, the consequences are different. For aIIb there is a moderate increase (+11%) in inter-subunit Hbonds and a small reduction in the global H-bonds network (-3.4%). However, for avb3 the mutation induces a dramatic increase (+41.5%) in inter-subunit H-bonds but a negligible decrease (-0.6%) in the H-bond global network (Table I). Overall, compared to the WT proteins, the P163S mutation induces a straightening of the inter-subunit domain that is slight with aIIbb3 but extensive with avb3. In the same way, global RMSD analyses of the complexes throughout 60 ns of molecular dynamics revealed b3 rearrangements that were modest with aIIb (black and green traces) and profound with av (red and blue traces) ( Figure S3).
In terms of individual bonds, in aIIbb3 the newly introduced S163 on b3 exchanges strong H-bonds with E168 on the asubunit. This contrasts with the WT complex where E168 exchanges H-bonds essentially with A263 on b3 and W110, P126 and F171 on aIIb all of which are lost in the presence of the mutation. Moreover, the new S163 now also exchanges H-bonds with R216 and L262 on b3. P163 does not appear in the WT Hbond list and neither do R216 and L262. P163 is also not in the Hbond list for WT avb3 where the introduced S163 now forms weak H-bonds with L262 and N156 on av. The main H-bonds in WT avb3 are between D259 (b3) and Y275 (av) S291 (b3) and E311 (av) and between T296 (b3) and L309 (av). In the presence of S163, the interaction between S291 (b3) and E311 (av) is much stronger while that between Y275 (av) and D259 (b3) is weaker. Several new interactions appear: S300 (b3) with D306 (av), D259 (b3) with Y221 (av) and Y166 (b3) with Y178 or E121 (av).
Major changes also occur for b3R261 that exchanges H-bonds with a number of amino acids on aIIb in the WT integrin: Y237, A95, F21, F419, W110, G170, F171 and Y288 while only forming H-bonds withY237 and F171 in the mutated form ( Figure S4). For av, the number of H-bonds involving b3R261 shows little change although they involve different partners: Y224, Y406, F278, Y406 and Y224 in the WT form; and Y406, F178, F159, Y224 and A96 in the presence of b3S163.

Summary of the Effects of the P163S Substitution
The major structural changes are highlighted when the WT and mutant forms of both aIIbb3 and avb3 are superimposed ( Figure 6). For avb3 note the unfolding of the small a-helix close to position 163 (between 169 and 174, lower yellow arrow, Figure 6B); a displacement towards the interface of the a-helix beginning at position 259 on b3 (yellow*) and the new fold appearing in the P163S mutant (between 166 and 174, yellow arrow) that projects toward the interface resulting in a clockwise rotation of the av subunit (yellow curved arrows). Comparison of the two forms of aIIbb3 ( Figure 6A) shows that the mutation only slightly displaces the small alpha-helix close to position 163 (lower yellow arrow) but results in the small alpha-helix beginning at position 259 (yellow*) moving away from the interface (approximately 23.5 Å ). This 3 10 -a-helix contains b3R261 that is now localized 3.5 Å outside of the aIIb headpiece when compared to the WT conformation. In contrast, b3R261 sinks deep into the bpropeller of the av headpiece (approximately 4.4 Å in comparison to the WT conformation).

Discussion
The crystal structure of the extracellular segment of integrin avb3 provided the first clear insights into the extracellular head domain structure and how conformation changes with the activation state of the integrin [14][15][16][17][18][19]. Close contacts between the two subunits primarily involved the av b-propeller and the b3 b-I (bA) domains. b-I also contains functionally important MIDAS and ADMIDAS sequences with 3 metal ion-binding domains. A key residue is b3R261 that lies at the core of the b-I domain-bpropeller interface and is surrounded by two concentric rings of predominantly aromatic a-subunit b-propeller residues. Sidechains of F21, F159, Y224, F278 and Y406 from the lower ring were said to interact with R261 directly. Residues Y18, W93, Y221, Y273 and S403 in the upper ring contact side-chains in the lower ring and provide a hydrophobic interface for residues flanking b3R261 in the so-called 3 10 -a-helix [14]. Additional contacts were also shown between more distant parts of the head domains of both subunits. It was noted even at this early time that b3P163, the amino acid mutated in our patient, lies in a loop adjacent to the 3 10 -helix of av.
Homology models were first used to extrapolate results for avb3 to aIIbb3 and predict contact interactions between aIIb and b3. These became redundant when a refined crystal structure of the complete aIIbb3 ectodomain obtained in the presence of Ca 2+ and Mg 2+ permitted direct analyses [16][17][18][19]. Water molecules that favor hydrogen bonding and metal coordination were located in the aIIbb3 but not the avb3 structure. Particularly highlighted were three non-conserved loop region structures (residues 71-85, 114-125 and 148-164) of human aIIb while K118 was said to form a salt bridge with E171 in the specificity-determining loop (SDL) of b3 (residues 159-188) that contains P163. The structural importance of these residues is highlighted by the large number of missense mutations in the b-propeller region of aIIb detected in patients with classic type I GT [2,3,27]. Crystallography also predicted that aIIb residues L116, K124 and R153 were close to one or more residues of the b3 SDL region. b3 residues I167, S168 and P169 were said to have a side chain or backbone within 5 Å of aIIb residues. Further proof for residues in close contact came from a cysteine substitution model that provoked the formation of disulfide-linked dimers when the mutated aIIb and b3 subunits were transfected into HEK 293 cells [16].
It is in this context that we now report a GT patient with a b3 P163S substitution with little or no expression of aIIbb3 at the platelet surface but with residual b3 and a usual presence of avb3 in her platelets. Transfection of WT and mutated integrins in CHO cells recapitulated the loss of cell surface expression of aIIbb3 in cells co-transfected with WT aIIb and b3S163, and confirmed the capacity of the mutated b3 to bind endogenous hamster av and form a heterodimer that was transferred to the cell surface. Notwithstanding, differences in surface expression of chimeric avb3 were observed (Figure 2) depending on the use of a monoclonal antibody to human b3, which detected similar levels of avb3 in cells transfected with the b3S163 variant compared to those transfected with WT b3, or a monoclonal antibody to human avb3, which showed a reduced expression between the WT avb3 and the avb3S163. The latter most likely reflects differences in the ability of hamster av to bind to WT b3 and b3S163 or to conformational changes within the epitope recognized by the LM609 antibody. For platelets of the patient,  avb3 had a mostly vesicular localization as previously described by us for normal platelets and for those of another type I GT patient with a homozygous ITGA2B E324K mutation and residual b3 [8]. The reason for this localization is unknown but is consistent with a trafficking role for avb3; roles for avb3 in transport of vitronectin and in the sensing of bacterial lipopeptides have been previously described [28,29]. As this was not a major thrust of our paper the localization of avb3 was not studied further. Significantly an adjacent b3S162L mutation was previously reported in a GT patient with much decreased amounts of platelet aIIbb3; S162 lies close to blade 2 of the propeller and its replacement by L162 results in unfavorable contacts at the aIIb and b3 interface underlining the structural importance of this particular b-I domain; avb3 was not studied by the authors [27,30].
Computer modeling and a molecular dynamics analysis confirmed that the P163S mutation affected the b3 interface with both av and aIIb and showed the advantages of the dynamic approach in evaluating the structural effects of amino acid substitutions. The previously detected salt bridge between aIIbK118 and b3E171 (16)(17)(18) was maintained at least partly in the WT integrin during the molecular dynamics run; but for the P163S mutant and due to the relative movements of the two subunits it was replaced by a salt bridge between b3E171 and aIIbR122. Globally, aIIbb3S163 showed a moderate 11% increase in intra-subunit H-bonds and a very weak decrease in the global H-bond network but avb3S163 showed a dramatic 41% increase in intra-subunit H-bonds without modifying the H-bond global network. Compared to the WT proteins, the P163S mutation induces a straightening of the inter-subunit interactions that is slight with aIIbb3 but extensive for avb3. These structural rearrangements result in positioning of b3R261 outside the bpropeller in aIIbb3 but deep inside for avb3. All in all, mutated avb3 appears to have an increased stability perhaps confirmed by the intensity of the residual b3 band observed in the patient's platelets by Western blotting.
Molecular dynamics simulations and modeling of aIIbb3 have recently been reported for a homozygous aIIb N2D mutation present in 4 siblings of an Israeli Arab family that affects blade 1 of the b-propeller [31]. There was no surface expression of aIIbb3 in platelets or after transfection of the mutated integrin in BHK cells; the mutated pro-aIIbb3 complex was formed but trafficking was impaired. N2 is surface exposed on the b-propeller and is highly conserved. Here, a H-bond between N2 and L366 of a calciumbinding domain in blade 6 of aIIb was disrupted, thereby impairing calcium binding essential for intracellular trafficking of pro-aIIbb3. When the equivalent mutation was introduced into avb3 it had a less deleterious effect in transfected BHK cells confirming a lower sensitivity of avb3 to calcium chelation. Molecular dynamic simulations of the wild-type and mutant proteins indicated that aa364-370 fluctuated more in the mutant aIIb with a shifting out of blade 6 [31].
Other mutations that differentially affect aIIbb3 and avb3 include a L196P mutation adjacent to the b3 MIDAS (amino acids 118-131) in two French GT patients that allowed residual (10 to 15%) expression of non-functional aIIbb3 [10,11]. Transfection of b3P196 with wild-type aIIb in CHO cells confirmed interference with aIIbb3 maturation yet avb3 was normally expressed [10]; a result similar to that now reported by us for b3P163S. A b3 L262P mutation gave residual aIIbb3 able to bind fibrin and with  platelets able to retract clots; yet the platelets did not bind Fg when stimulated [32]. Leu262 occurs within an intrachain disulfide loop (between C232 and C273) important for subunit assembly and is joined to b3R261 in the 3 10 -a-helix. When transiently transfected with wild-type aIIb in COS-7 cells, aIIbb3P262 allowed normal heterodimer formation but export from the endoplasmic reticulum was delayed and those complexes that reached the surface were unstable. b3P262 transfected in human embryonic kidney 293 cells formed a complex with av and retracted fibrin clots although the cells did not interact with immobilized Fg. As we have reviewed elsewhere, other mutations within b3 mimic b-I domain P163S by differently affecting aIIbb3 and avb3 expression [33]. These include breakage of some of the 56 disulfides in the EGF domains of b3 [13,[34][35][36]. For example, disrupting C473-C503 caused reduced surface expression of avb3 relative to aIIbb3 whereas disruption of C437-C457 by C457S resulted in a significant reduction of aIIbb3 compared to avb3 [13]. Molecular dynamics analysis was performed using a mutated b3 fragment composed of the four EGF domains and b-tail domain derived from aIIbb3 and avb3 crystal structures [13]. The mutated aIIbb3 structure was changed considerably from the native one and was stable in a new activated conformation whereas the final avb3 structure resembled the starting conformation.
Another mutation in b3 exerting a more deleterious effect on aIIbb3 than avb3 expression is H280P (variant Osaka-5). H280P was found in three unrelated Japanese patients (one homozygous and two heterozygous) with residual aIIbb3 expression [9,37]. Platelets expressed about half the normal amounts of avb3 whereas aIIbb3 levels were reduced to about 6%.
Taken in this context, our studies on b3 P163S provide new evidence as to how missense mutations within the extracellular domain of b3 can differentially influence aIIbb3 and avb3 expression. We show how b3S163 affects the three-dimensional structure of the integrins differently and that avb3 can even become more stable. This has important implications for considering genotype/phenotype relationships in Glanzmann thrombasthenia. Up-to-now, no clear differences in phenotype have been reported between patients with ITGA2B or ITGB3 mutations [2,3,38]. However, the structural consequences of ITGB3 missense mutations are clearly variable and therefore it is necessary to establish for each patient how aIIbb3 and avb3 are affected. In this respect, the human disease differs from mouse models where the Itgb3 gene is specifically deleted [2]. Figure S1 Flow cytometry measuring the binding of selected monoclonal antibodies to platelets of the patient. This study was performed according to our standard procedures using a Becton Dickenson FACScan [39,40]. Note the minimal binding of AP2 (anti aIIbb3) and Tab (anti-aIIb); a slightly higher binding of AP3 (anti-b3) and a normal binding of BX1 (anti-GPIba) to the platelets of the patient. (TIF) Figure S2 Conservation of b3 Pro163. Residue P163 (*) of b3 is highly conserved within mammals and vertebrates (A) and within different integrin b-subunits in man (B). Also shown is the highly conserved nature of aIIb amino acids (C) and of av amino acids (D) forming H-bonds with b3P163. In dotted boxes are amino acids participating in H-bonds within aIIbb3 but not within avb3.

Supporting Information
(TIF) Figure 6. Summary of the major changes seen in the molecular dynamics runs. Position 163 on b3 is shown as red spheres. A/ Superimposition of the two forms of aIIbb3: aIIb associated with WT b3 is in silver glass ribbon while blue ribbon highlights aIIb in complex with the b3P163S mutant; orange ribbon denotes b3. Note that the mutation induces only a slight displacement of the small a-helix close to position 163 (yellow arrow below) and a larger change for the small a-helix beginning at position 259 but outside of the interface (yellow*). B/Superimposition of the two forms of avb3: av associated with WT b3 is in silver glass ribbon while blue ribbon shows aIIb associated with the b3P163S mutant; orange ribbon denotes b3. Note the unfolding of the small a-helix close to position 163 (yellow arrow below), the large displacement towards the interface of the a-helix beginning at position 259 on b3 (yellow*) and the new fold appearing in the P163S mutant (yellow arrow) that is projected towards the interface resulting in a clockwise rotation of the av sub-unit (yellow curved arrows). Windows correspond to a zoom of the regions marked by the asterisk. doi:10.1371/journal.pone.0078683.g006 Figure S3 Molecular dynamics analysis. Plots of RMSD vs. time of the global integrin complex of the av and b3 subunit headpieces during a complete (60 ns) molecular dynamics run. Shown are the results for wild-type aIIbb3 and avb3 and for aIIbb3S163 and avb3S163. (TIF) Figure S4 3D-modelisation of amino acids interacting with b3. Amino acids are represented as sticks; b3R261 is coloured in magenta while amino acids from aIIb or avb3 are coloured in dark green for the wild type integrin and in pink and light green for the mutated form. The initial position for b3R261is superimposed as a transparent image. (TIF) Case History S1.