Blood, Vol. 90 No. 8 (October 15), 1997:
pp. 3082-3088
A Leu117 
Trp Mutation Within the RGD-Peptide Cross-Linking Region of 
3 Results in Glanzmann Thrombasthenia by Preventing 
IIb3 Export to the Platelet Surface
By
Ramesh B. Basani,
Deborah L. Brown,
Gaston Vilaire,
Joel S. Bennett, and
Mortimer Poncz
From the Departments of Pediatrics and Medicine, The University of Pennsylvania School of Medicine, Philadelphia, PA.
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ABSTRACT |
We report a case of Glanzmann thrombasthenia in a Pakistani child whose platelets express less than 10% of the normal amount of 
IIb
3 on their surface. Single-stranded conformation polymorphism analysis of the exons of the patient's IIb and 3 genes showed an abnormality in exon 4 of the 3 gene. Direct sequence analysis showed that the patient was homozygous for a T 
G nucleotide substitution in this exon, resulting in the replacement of a highly conserved Leu at position 117 with Trp. Heterologous expression of IIb3 containing the 3 mutation in COS-1 cells confirmed the pathogenicity of the Leu117 Trp substitution and showed that it resulted in the intracellular retention of malfolded IIb3 heterodimers. Additional site-directed mutagenesis at position 117 indicated that, although the smaller hydrophobic amino acid Val could be substituted for the wild-type Leu, the larger hydrophobic amino acids Trp and Phe or the charged amino acids Asp and Lys were not tolerated. These studies indicate that Leu117 in 3 plays a critical role in attaining the correct folded conformation of IIb3. These studies also suggest that the hydrophobic side chain of Leu117 is likely folded into the interior of 3, where it serves to stabilize internal packing of the protein and determines its overall shape.
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INTRODUCTION |
PLATELET AGGREGATION is responsible for primary hemostasis and results from the cross-linking of activated platelets by fibrinogen and/or von Willebrand factor bound to the integrin IIb3 (GPIIb-IIIa, CD41CD62).1,2 Consequently, inherited abnormalities in either the quantity or function of IIb3 result in the bleeding disorder Glanzmann thrombasthenia.3,4 More than 30 mutations that result in the thrombasthenic phenotype have been identified in either IIb or 3.5-21 Although some of these mutations have been complex gene rearrangements, gene deletions, mRNA splicing abnormalities, frameshifts, or missense mutations that prevent IIb or 3 synthesis,5-12 others have been single amino acid substitutions or small deletion or truncations.13-21 The latter have proven to be particularly instructive in understanding IIb3 biology.
Congenital or site-directed mutations involving the region of IIb that contains its four putative calcium-binding domains6,18-20,22 result in a distinctive phenotype. These mutations neither destabilize IIb nor prevent the assembly of IIb3 heterodimers. However, assembled heterodimers containing the IIb mutations are not recognized by heterodimer-specific monoclonal antibodies (MoAbs) and are retained intracellularly, presumably because they are malfolded. In contrast, congenital or site-directed mutations involving a region of 3 extending from residues 119 to 130 do not impair IIb3 expression, although the more proximal mutations impair fibrinogen binding to IIb3.23
We report here studies of a patient with Glanzmann thrombasthenia whose platelets express little, if any, IIb3 on their surface and who is homozygous for a Leu Trp substitution at 3 residue 117. Unlike previously described mutations in this region of 3, the phenotype resulting from the Leu117 Trp mutation is identical to that of mutations involving the IIb calcium binding region. Because both the 3 Leu117 Trp and the IIb mutations prevent recognition of IIb3 by heterodimer-specific MoAbs, it is possible that the affected regions of 3 and IIb are physically associated in the intact IIb3 heterodimer and form the epitopes recognized by these antibodies. Furthermore, because a notable feature of these antibodies is to inhibit fibrinogen binding to IIb3, it is also possible that the affected regions of 3 and IIb constitute the IIb3 ligand binding site.
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MATERIALS AND METHODS |
Case report.
Patient MK, a Pakistanian child, presented at 1 day of age with petechiae, purpura, and a platelet count of 181,000/µL. When the petechiae recurred at 4 weeks of age, a workup showed absence of platelet aggregation in response to adenosine diphosphate (ADP), epinephrine, and collagen, but a normal response to ristocetin. Blood samples for the studies described below were obtained from the patient and her parents after informed consent. The studies were approved by the Human Subjects Internal Review Committee at the Children's Hospital of Philadelphia.
Flow cytometry.
Flow cytometry was performed using a panel of MoAbs conjugated to florescein isothiocyanate (FITC) as described previously.16 The MoAbs used for these studies were A2A9, an MoAb that interacts with an epitope expressed on the extracellular domain of the intact IIb3 heterodimer24,25; B1B5, an MoAb that recognizes an epitope located on the extracellular portion of the 18 nm tail of IIb proximal to its transmembrane domain25; SSA6, an MoAb that recognizes an epitope located on the extracellular portion of the 18 nm tail of 3 just proximal to its transmembrane domain25; PAC-1, an MoAb that recognizes an epitope located on the extracellular portion of IIb3 and expressed exclusively by its activated conformation26; and AP-1, an MoAb specific for platelet GPIb and a gift of Dr Thomas Kunicki (Scripps Research Institute, La Jolla, CA).27 Measurements of PAC-1 binding were performed after stimulating platelets with 0.2 µmol/L phorbol myristate acetate for 5 minutes at 25°C.
Identification of the thrombasthenic mutation.
Genomic DNA containing all of the exons of the IIb and 3 genes and 500 bp of DNA immediately upstream of each transcriptional start site was isolated from the patient and from normal controls, as previously described.28 Screening for mutations was performed using single-stranded conformation polymorphism analysis (SSCPA)
.29,30 Polymerase chain reaction (PCR) amplification was performed in a 100 µL reaction volume containing 500 ng of genomic DNA, 100 µmol/L dNTPs, 1 µL of 32P-dCTP (10 mCi/mL; Dupont, NEN Research Products, Boston, MA), 200 ng of each primer, and 1 U Taq polymerase. Thirty cycles of PCR amplification were performed as follows: denaturation at 94°C for 15 seconds, annealing at 60°C for 30 seconds, and elongation at 72°C for 40 seconds. Equal quantities of the PCR products were used for SSCPA. The PCR products in 95% formamide were heated for 2 minutes at 95°C and loaded onto a 35 × 42.5 cm, 6% DNA sequencing polyacrylamide gel (IBI/VWR Scientific, Bridgeport, NJ) containing no urea. Electrophoresis was performed at 30 watts and 8°C for 2 hours. The completed gels were dried and examined by autoradiography.
DNA fragments that migrated abnormally in the SSCPA gel were directly sequenced using the fmol DNA Cycle Sequencing Kit (Promega, Madison, WI) in a BIOS Oven III thermal cycler (BIOS Laboratories, New Haven, CT), as previously described.16,20 DNA fragments were also subcloned using the commercial TA Cloning Kit (Invitrogen Co, San Diego, CA) and sequenced using Sp6 and T7 primers and a commercial Sequenase sequencing kit (US Biochemicals, Cleveland, OH).
Heterologous expression of recombinant IIb3.
To examine their effect on IIb3 assembly and intracellular transport, identified mutations were introduced into the corresponding wild-type sequence using an overlap PCR technique, as previously described.31 PCR amplification was then performed using VENT polymerase (Promega) to decrease the frequency of PCR-induced mutations.32 The resulting PCR products were digested with the endonucleases BstBI (NEB, Inc, Beverly MA) and Mlu I and subcloned into similarly digested IIb or 3 cDNA in PUC18 (GIBCO/BRL, Gaithersburg, MD). After sequencing to insure the fidelity of the PCR reaction, the DNA was shuttled into the expression vector pMT2ADA.33 Site-directed mutations in IIb or 3 were produced using the same mutagenesis strategy.
Recombinant IIb3 was expressed in vitro using COS-1 cells, as previously described.16,20 Briefly, COS-1 cells were cotransfected with cDNAs for IIb and 3 by the lipofection method (Lipofectin Reagent; GIBCO/BRL).16 Forty-eight hours after transfection, the cells were either metabolically labeled with 35S-methionine (Dupont) at 200 µCi/mL for 60 minutes16 or surface-labeled with biotin using 5 mmol/L N-hydroxysulfosuccinimide (NHS-LC) biotin phosphate-buffered saline (PBS) (Pierce, Rockford, IL) for 30 minutes at room temperature.18 The cells were then extracted with 0.02 mol/L Tris-HCl buffer, pH 7.2, containing 1% Triton X-100 and IIb3 was immunoprecipitated using SSA6 or A2A9. To identify proteins on the COS-1 cells surface, biotinylated immunoprecipitated proteins were electrophoresed on a 7% sodium dodecyl sulfate-polyacrylamide gel and transferred to an Immobilon membrane (Millipore, Bedford, MA). The resulting blot was blocked with bovine serum albumin and Tween-20 (Sigma, St Louis, MO) and incubated with 1:4,000 dilution of streptavidin-horseradish peroxidase (Amersham, Arlington Heights, IL) for 1 hour at room temperature. Biotinylated proteins were then identified using the ECL kit (Amersham) according to the manufacturer's instructions.
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RESULTS |
Quantitation of IIb3 on propositus platelets.
The inability of platelets from patient MK to aggregate in response to ADP, epinephrine, or collagen suggested that she suffered from Glanzmann thrombasthenia. To confirm this diagnosis, we measured the level of IIb3 on the surface of her platelets by flow cytometry. As shown in Fig 1, when compared with platelets from a normal control, MK's platelets expressed less than 10% of the normal amount of IIb-3 when they were stained with the IIb-specific MoAb B1B5 and the IIb3-specific MoAb A2A9, and there was little or no staining with the activation-dependent, IIb3-specific MoAb PAC-1. In contrast, MK's platelets bound a normal amount of the anti-GPIb MoAb AP-1, confirming that the defect in the patient's platelets was confined to IIb3.
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| Fig 1.
Measurement of the IIb3 content of patient platelets using flow cytometry. Comparison of IIb-3 expression on the surface of patient and control platelets using the IIb-specific MoAb B1B5, the IIb3 heterodimer-specific MoAb A2A9, the activation-dependent anti-IIb3 MoAb PAC-1, and the GP1b-complex specific MoAb AP-1. The data in the figure are expressed as the percentage of antibody binding to patient versus control platelets. Measurements of PAC-1 binding were made after stimulating platelets with 0.2 µmol/L phorbol myristate acetate. ( ) Control; ( ) patient.
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Identification of mutations responsible for the patient's thrombasthenia.
To identify the mutation or mutations responsible for the patient's thrombasthenia, genomic DNA from the patient was screened using SSCPA and oligonucleotide primers designed to synthesize DNA fragments containing portions of each exon of the IIb and 3 genes and the 500 bp immediately upstream of each gene's transcriptional start site.34-36 As shown in Fig 2A, the SSCPA of exon 4 of MK's 3 gene showed an abnormal faster migrating band. Direct cycle sequence analysis of PCR products from the patient and a normal control showed that the patient's DNA was homozygous for a T G nucleotide substitution at nucleotide 445 resulting in a Leu117 Trp mutation (Fig 2B). To confirm that MK was homozygous at this position, her PCR product was subcloned into a TA cloning vector and individual clones were sequenced. Of the six clones sequenced, all had the T G mutation (data not shown).
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| Fig 2.
Analysis of the patient's 3 gene. (A) SSCP analysis of 3 exon 4 in a normal control (lane 1), four unrelated thrombasthenic patients (lanes 2, 3, 5, and 6), and the patient (lane 4). The patient's lower band migrated faster than any of the other studied samples. (B) Sequence analysis of wild-type (WT) and the patient's (MK) DNA for 3 exon 4 showing a single T G substitution in this exon.
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Expression of the mutant 3 allele in vitro.
To confirm that the 3 Leu117 Trp amino acid substitution was responsible for the patient's thrombasthenic phenotype, we introduced this mutation into the sequence of a wild-type 3 cDNA and expressed the mutant in COS-1 cells, either alone or with wild-type IIb. Although the mutation did not demonstrably impair 3 synthesis (Fig 3A) or prevent the assembly of IIb3 heterodimers (Fig 3B), the assembled heterodimers were not recognized by the MoAb A2A9 (Fig 3C), suggesting that the heterodimers were malfolded. To determine whether the abnormal folding affected IIb3 stability, pulse-chase studies were performed. As shown in Fig 3D, the presence of 3 Leu117 Trp had no obvious effect on the stability of IIb3 over the 8-hour chase period.
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| Fig 3.
Expression of 3 containing the Leu117 Trp substitution in COS-1 cells. Wild-type (WT) and mutant (MK) 3 were expressed in COS-1, either alone or with IIb. Cells were then either metabolically labeled with 35S-methionine or surface labeled with biotin-labeled proteins were immunoprecipitated by the antibodies indicated below and examined by SDS-polyacrylamide gel electrophoresis. (A) Wild-type and mutant 3 were expressed alone in COS-1 cells and immunoprecipitated from cells labeled with 35S-methionine using the 3-specific MoAb SSA6. (B and C) COS-1 cells were cotransfected with IIb and either wild-type or mutant 3. After labeling the cells with 35S-methionine, IIb-3 was immunoprecipitated using the 3-specific MoAb SSA6 (B) or the heterodimer-specific MoAb A2A9 (C). (D) The cells were treated as in (B), but were pulse-chased for the various time intervals indicated. (E) The surface of COS-1 cells cotransfected with IIb and either wild-type or mutant 3 was biotin labeled and immunoprecipitated using SSA6. WT is the wild-type complex, MK refers to IIb-3Leu117  Trp complexes, and JF refers to IIbGly273 Asp-3 complexes. Pro-IIb corresponds to the single-chain IIb precursor; IIb- corresponds to the IIb heavy chain.
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It is noteworthy that throughout the chase IIb was not cleaved into heavy and light chains (Fig 3D). Although this suggests that the mutant heterodimers were not exported from the endoplasmic reticulum to the Golgi complex in which IIb cleavage occurs,37 IIb cleavage is not required to express IIb3 on the cell surface. Thus, to confirm that 3 Leu117 Trp actually prevents the export of IIb3 to the cell surface, we biotinylated the surface of COS-1 cells expressing wild-type IIb3, IIb3 containing 3 Leu117 Trp, and IIb3 containing IIb Gly273 Asp, a mutation known to impair the export of IIb3 to the cell surface.16 We then immunoprecipitated IIb3 using the 3-specific MoAb SSA6. Although labeled complexes were present on the surface of cells expressing wild-type IIb3, none were not present on the surface of cells expressing either of the IIb3 mutants (Fig 3E).
Effect of other mutations at 3 position 117 on the expression of IIb3.
The 3 mutation detected in patient MK resulted in the replacement of a highly conserved nonpolar Leu with a nonpolar Trp residue. To determine if the consequences of this mutation were due to the introduction of the bulky size indole side chain of Trp at this location in 3, we introduced other selected amino acids into position 117 by site-directed mutagenesis and examined their effect on IIb3 expression in COS-1 cells. Phe and Val were selected as examples of nonpolar hydrophobic amino acids with differently sized side chains. The charged amino acids Asp and Lys were also selected because Leu117 precedes an array of charged and polar residues that have been implicated in both ligand and divalent cation binding to IIb3.23 As shown in Fig 4A and B, the presence of Trp, Asp, Lys, Phe, or Val has no apparent effect on 3 synthesis (Fig 4A) or on the ability of 3 to associate with IIb (Fig 4B). However, only heterodimers containing the wild-type Leu or Val at 3 position 117 could be immunoprecipitated using A2A9 (Fig 4C). Thus, these experiments indicate that, although a nonpolar residue is required at position 117 for proper 3 folding, there are limits to the size of the side chain of the residue that can be tolerated. Moreover, the presence of a charged residue at this position is not tolerated, regardless of the size of its side chain.
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| Fig 4.
Expression of 3 containing additional 3117 substitutions in COS-1 cells. Transient expression studies in COS-1 cells were performed using either 3 mutants alone (A) or in combination with wild-type IIb (B and C). In (A) and (B), the recombinant proteins were immunoprecipitated with SSA6 and in (C) with A2A9. The specific mutation introduced at 3117 is indicated for each lane. WT is the wild-type complex. Pro-IIb corresponds to the single-chain IIb precursor; IIb- corresponds to the IIb heavy chain.
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DISCUSSION |
The basis for the Glanzmann thrombasthenia of the patient we studied was a Leu117 Trp mutation in the 3 subunit of the integrin IIb3. Although the mutation did not impair 3 synthesis or the assembly of IIb3 heterodimers, it prevented export of the assembled heterodimers to the surface of transfected COS cells, presumably by causing their retention in a pre-Golgi compartment.38 We have observed that the ability to express IIb3 on the COS cell surface is a sensitive index of the integrity of IIb3 folding.22 Thus, it is likely that the impairment of IIb3 expression by the Leu117 Trp mutation results from a mutation-induced change in the conformation of the assembled heterodimer, a conclusion consistent with the failure of the mutant heterodimers to be recognized by the heterodimer-specific MoAb A2A9.
Leu117 is located near the proximal end of an array of polar and charged amino acids extending from Asp113 to Ser130 that has been implicated in ligand binding to IIb339 and in low-affinity association of divalent cations with 340 (Fig 5). Furthermore, the region around amino acid 117 is highly conserved in the 3 of various species and in other subunits such as 1 and 2.41 Based on homology modeling, it has been proposed that this region of 3 adopts a conformation similar to that of a metal ion-dependent adhesion site or "MIDAS" motif.42 However, in the absence of actual structural information about this region of 3, it is only possible to speculate about the structural consequences of mutations at position 117. Nevertheless, it is reasonable to assume from considerations of protein stability that the hydrophobic aliphatic side chain of Leu117 in this environment would be folded into the interior of 3. The inability to tolerate charged Asp or Lys residues at this position supports this assumption.43 Furthermore, there appears to be substantial steric constraint imposed on the folding of 3 at this position because the smaller side chain of Val, but not the bulky hydrophobic side chains of Trp and Phe, could be tolerated. In contrast, replacement of Asp119, just two residues carboxyl terminal to Leu117, with Tyr (the Cam 3 variant44 ) or alanine23 or replacement of Ser121, Ser123, Asp126, Asp127, and Ser130 with alanine23 neither affected IIb3 expression nor the ability of the heterodimer to interact with the complex-specific MoAb 10E5. Identical results were observed in studies of two naturally-occurring mutations involving the Arg214 (Arg214 tryptophen15 and Arg214 glutamine14 ). Thus, the loss of charged or polar residues from these regions of 3 does not appear to significantly affect the folding of the protein. Because the side chains of these residues likely project from the exterior surface of 3 into the aqueous milieu, their presence would not be involved in the interior packing that appears to be critical for the final 3 conformation. This formulation is consistent with previous studies of the effect of amino acid substitutions on protein stability.43 These studies suggest that a only a limited number of residues in a protein contribute significantly to its folded structure. For example, it was observed in studies of the dimer interface of the DNA-binding domain of the 
repressor that the ability to tolerate amino acid substitutions correlated roughly with the accessibility of the wild-type side chain to solvent.45 Moreover, replacement of a major contributor to the hydrophobic core of T4 lysozyme, Ile 3, by Trp or Tyr had a substantial deleterious effect on lysozyme stability, as did the introduction of the polar or charged amino acids Ser, Thr, and Asp.46
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| Fig 5.
Region of 3 affected by the Leu117 Trp mutation. The amino acid sequence of the region of 3 extending from residues 113 to 130 is shown in the single letter code. Leu117 is designated by the darker arrow and Asp119 by the lighter arrow. The polar and charged amino acids mutated in Bajt and Loftus23 are underlined.
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The consequences of the Leu117 Trp mutation in 3 identified in this patient are the same as those of four mutations involving the putative calcium binding region of IIb.16,18-20 Like Leu117 Trp, these mutations neither impair IIb synthesis nor the assembly of IIb3 heterodimers, but inhibit IIb3 export to the cell surface. Moreover, they also specifically impair the recognition of IIb3 by heterodimer-specific MoAbs, suggesting the possibility that the regions of IIb and 3 containing these mutations physically interact to form the epitopes for these antibodies. Previous studies mapping the ligand binding region of IIb3 with fibrinogen peptides support this possibility. It was found that peptides containing the sequence RGD, present at two places in the fibrinogen chain, cross-link to a region of 3 encompassing amino acids 109-171,39 whereas peptides corresponding to the carboxyl terminus of the fibrinogen 
chain cross-link to a region of IIb corresponding to its second putative calcium binding domain.47 Although both classes of peptides inhibited ligand binding to IIb3, they did so in a mutually exclusive manner,48 suggesting that the ligand binding site on IIb3 consists of a surface containing of segments of both IIb and 3. A similar conclusion was reached by Loftus et al,49 who also found that the amino terminal third of IIb defined the ligand specificity of IIb3. It is noteworthy that 3 Leu117 Trp and the IIb calcium binding domain mutations reside either within or in proximity to the peptide cross-linking regions. Thus, it is possible that these mutations affect the conformation of the ligand binding surface, thereby explaining their effect on heterodimer-specific MoAb binding and supporting the notion that the segments of IIb and 3 containing the mutations are physically associated.
In summary, we have identified a naturally occurring 3 mutation, Leu117 Trp, which likely affects the conformation of IIb3 and results in the intracellular retention of mutant heterodimers. Although Leu117 Trp is located in immediate proximity to the previously described Cam mutation, Asp119 Tyr, its consequences are markedly different because the latter had no effect of IIb3 expression, but rather prevented ligand binding to the heterodimer. Thus, our results emphasize that the consequences of individual mutations in IIb and 3 mutations, regardless of their location in the IIb or 3 sequence, can be unique and are ultimately determined by their effect on subunit and heterodimer folding.
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FOOTNOTES |
Submitted February 25, 1997;
accepted June 16, 1997.
Supported in part by Grant No. HL40387 (to J.S.B. and M.P.), a grant from the Schulman Foundation (M.P.), and a grant from The Council for Tobacco Research-USA, Inc (#3152 to M.P.).
Address reprint requests to Mortimer Poncz, MD, The Children's Hospital of Philadelphia, 34th St and Civic Center Blvd, Philadelphia, PA 19104.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hearly marked
``advertisment'' in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
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ACKNOWLEDGMENT |
The authors thank Drs Simon and Margaret Karpatkin for generously sharing the patient presented in this report.
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Abstract
Introduction
Abstract