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Blood, Vol. 96 No. 1 (July 1), 2000: pp. 161-169

HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY

A Leu262Pro mutation in the integrin beta 3 subunit results in an alpha IIb-beta 3 complex that binds fibrin but not fibrinogen

Christopher M. Ward, Anita S. Kestin, and Peter J. Newman

From the Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, WI; the Department of Medicine, Brown University School of Medicine, Providence, RI; and the Departments of Cellular Biology and Pharmacology, Medical College of Wisconsin, Milwaukee, WI.


    Abstract
Top
Abstract
Introduction
Patients and methods
Results
Discussion
Acknowledgments
References

Platelet retraction of a fibrin clot is mediated by the platelet fibrinogen receptor, alpha IIbbeta 3. In certain forms of the inherited platelet disorder, Glanzmann thrombasthenia (GT), mutant alpha IIbbeta 3 may interact normally with fibrin yet fail to support fibrinogen-dependent aggregation. We describe a patient (LD) with such a form of GT. Platelets from LD supported normal clot retraction but failed to bind fibrinogen. Platelet analysis using flow cytometry and immunoblotting showed reduced but clearly detectable alpha IIbbeta 3, findings consistent with type II GT. Genotyping of LD revealed 2 novel beta 3 mutations: a deletion of nucleotides 867 to 868, resulting in a premature stop codon at amino acid residue 267, and a T883C missense mutation, resulting in a leucine (Leu) 262-to-proline (Pro) substitution. Leu262 is highly conserved among beta  integrin subunits and lies within an intrachain loop implicated in subunit association. Leu262Probeta 3 cotransfected with wild-type alpha IIb into COS-7 cells showed delayed intracellular maturation and reduced surface expression of easily dissociable complexes. In human embryonic kidney 293 cells, Leu262Probeta 3 formed a complex with endogenous av and retracted fibrin clots similarly to wild-type beta 3. The same cells, however, were unable to bind immobilized fibrinogen. The molecular requirements for alpha IIbbeta 3 to interact with fibrin compared with fibrinogen, therefore, appear to differ. The region surrounding beta 3 Leu262 may maintain beta 3 in a fibrinogen-binding, competent form, but it appears not to be required for receptor interactions with fibrin. (Blood. 2000;96:161-169)

© 2000 by The American Society of Hematology.


    Introduction
Top
Abstract
Introduction
Patients and methods
Results
Discussion
Acknowledgments
References

The aggregation of platelets and subsequent thrombus formation is mediated by the platelet-specific integrin alpha IIbbeta 3 (glycoprotein IIb-IIIa). Platelet alpha IIbbeta 3 is a heterodimeric surface receptor consisting of an alpha (alpha IIb) and a beta (beta 3) subunit in a noncovalent, cation-dependent complex.1 Platelet aggregation depends on alpha IIbbeta 3 binding of fibrinogen,2 but the receptor can recognize other ligands, including fibronectin, von Willebrand factor (vWf), and vitronectin.3 alpha IIbbeta 3 binding of soluble fibrinogen requires activation of the receptor by extracellular or intracellular events, a process that is poorly understood. A valuable tool for studying alpha IIbbeta 3 structure and function is the rare, autosomal recessive bleeding disorder Glanzmann thrombasthenia (GT), in which alpha IIbbeta 3 is dysfunctional or absent.4

GT mutations characterized at the molecular level are heterogenous and affect the alpha IIb and beta 3 genes equally.4,5 In many patients, gene deletions, frameshifts, and premature terminations result in alpha IIbbeta 3 expression at less than 5% of normal levels (type I GT). Of more interest are cases in which a dysfunctional alpha IIbbeta 3 complex is present on the platelet surface at reduced (type II GT) or nearly normal (variant GT) levels. Type II and variant GT are predominantly due to point mutations of the beta 3 gene in 2 critical areas. First, mutations of the charged residues arginine (Arg) 119, Arg214, and Arg2164 in the beta 3 extracellular domain lead to the identification of a cation binding motif (DXSXS), which is critical for ligand binding and homologous to the metal ion-dependent adhesion site (MIDAS) of certain integrin alpha  subunits.6,7 Second, mutations in the cytoplasmic domain of beta 3, such as Arg724Stop,8 result in a receptor that cannot be activated by intracellular signals (inside-out signaling) but still binds ligand in response to extracellular events. Therefore, thrombasthenic mutations can identify critical residues in alpha IIbbeta 3 that participate in ligand binding and receptor activation.

The molecular events involved in alpha IIbbeta 3 ligand binding have been partly described; like many integrins, alpha IIbbeta 3 recognizes an Arg-glycine (Gly)-aspartic acid (Asp), or RGD, motif present in some adhesive ligands.3 One of these, fibrinogen, is an oligomeric complex of 3 paired subunits, the Aalpha , Bbeta , and gamma  chains, and contains 4 RGD motifs, 2 in each Aalpha chain, at residues 95 to 97 and 572 to 574.9 In addition, a 12-amino acid motif, HHLGGAKQAGDV, comprising residues 400 to 411 of the gamma  chain, binds alpha IIbbeta 3 and competes with RGD for binding.10 Studies with fibrinogen mutants have shown that this C-terminal dodecapeptide is essential for fibrinogen-dependent platelet aggregation, whereas the Aalpha -chain RGD sequences are not required.11-13 Several research groups have attempted to localize the fibrinogen-binding site at the receptor level. Recombinant truncated extracellular domains of alpha IIb (residues 1-233) and beta 3 (residues 111-318) formed an RGD-binding complex.14 Cross-linking and peptide studies identified beta 3 residues 109 to 171 and 211 to 222 as potential fibrinogen-binding sites.15-17 These 2 regions include residues identified in GT or by mutagenesis6 as part of a MIDAS-like cation binding site in beta 3. In alpha IIb, the gamma -chain peptide of fibrinogen was cross-linked to residues 294 to 314 of alpha IIb, suggesting that this is also a ligand-recognition site,18 and mutation of alpha IIb residues Gly184 to Gly193 was reported to abolish fibrinogen binding.19

The molecular interactions of alpha IIbbeta 3 with insoluble fibrin are less well understood. During clot retraction, activated platelets cause a marked reduction in the volume of a fibrin clot.20 Clot retraction can also be induced by nucleated cells21 and is integrin dependent.22 In particular, the beta 3 subunit is important in binding fibrin, triggering retraction through links between the cytoplasmic domain of beta 3 and cytoskeletal proteins.23 In cultured mammalian cell lines, beta 3, as part of the vitronectin receptor, alpha vbeta 3, induced spontaneous fibrin-clot retraction, whereas alpha IIbbeta 3 required prior activation of the integrin for retraction.24,25 In contrast to the situation with platelet aggregation, the C-terminal of the fibrinogen gamma  chain does not appear to be required for fibrin-clot retraction mediated by alpha IIbbeta 3,12,13 suggesting that fibrinogen and fibrin may bind to different sites on the receptor.

In the current study, we identified a novel beta 3 mutation, leucine (Leu) 262 to proline (Pro) (Leu262Pro), in a patient with type II GT whose platelets showed normal clot retraction. This mutation is predicted to alter the conformation of a region of beta 3 located between 2 putative ligand-binding sites and involved in subunit interactions. By expressing Leu262Probeta 3 in complex with alpha v in mammalian cells, we demonstrated that the mutant receptor binds fibrin in a similar manner to wild-type beta 3 but does not recognize fibrinogen.


    Patients and methods
Top
Abstract
Introduction
Patients and methods
Results
Discussion
Acknowledgments
References

Patient studies

Patient LD was a 3-year-old girl who presented postnatally with skin petechiae and ecchymoses. She had frequent bruising with minimal trauma and gum bleeding after tooth eruption. The patient's mother had a history of menorrhagia but was found to have von Willebrand disease; her father had no history of abnormal bleeding. Laboratory testing of LD showed that she had a normal platelet count (360 × 109/L) and a prolonged bleeding time. Prothrombin time, partial thromboplastin time, factor VIII, vWF antigen, and ristocetin cofactor levels were also normal.

Clot retraction by platelets

Whole blood from patient LD, her parents, and healthy controls was anticoagulated with acid citrate dextrose A, and prostaglandin E1 (50 ng/mL) was added before centrifugation at 220g to separate platelet-rich plasma (PRP). PRP samples were diluted in homologous platelet-poor plasma to final platelet counts of 2.5, 1.25, and 0.6 × 108 platelets/mL and incubated in glass tubes for 5 minutes at 37°C. Clot formation was induced by calcium chloride (8 mmol/L final concentration), and samples were photographed after 1 hour of incubation at 37°C.

Antibodies

The alpha IIb-specific monoclonal antibody (mAb) Tab was a gift from Dr Rodger McEver (University of Oklahoma).26 The beta 3-specific mAb AP3 was described previously.27 The mAb AP1, which recognizes GPIb, and the mAb AP2, which binds to a complex-dependent epitope on alpha IIbbeta 3,28 were provided by Dr Robert R. Montgomery (Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, WI). The mAb LM609, which recognizes the alpha vbeta 3 complex,29 was a gift from Dr David A. Cheresh (Scripps Institute, La Jolla, CA). The mAb P4C10 against human beta 1 integrin was obtained from GIBCO BRL (Gaithersburg, MD). Rabbit polyclonal anti-alpha IIb and anti-beta 3 antibodies were prepared at the Blood Research Institute by using standard procedures.

Flow-cytometric analysis of platelet membrane glycoproteins

PRP from patient and control samples was centrifuged at 500g, and the platelet pellet was washed and resuspended at 4 × 108 platelets/mL in RCD-EDTA buffer (108 mmol/L sodium chloride [NaCl], 38 mmol/L potassium chloride, 1.7 mmol/L sodium bicarbonate, 21.2 mmol/L sodium citrate, 27.8 mmol/L glucose, 1.1 mmol/L magnesium chloride-6H20 , and 2 mmol/L EDTA [pH 6.5]). For each analysis, 1 × 107 platelets in RCD-EDTA buffer plus 0.2% (wt/vol) bovine serum albumin (BSA) were incubated with a mouse mAb at a final concentration of 20 µg/mL for 1 hour at room temperature. Samples were then washed in the same buffer and incubated with a 1:20 dilution of fluorescein isothiocyanate-conjugated-labeled goat-antimouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA) for 30 minutes in the dark. The samples were washed again, diluted in RCD-EDTA buffer and analyzed on a fluorescence-activated cell-sorter scan flow cytometer (Becton Dickinson, Mountain View, CA).

Semiquantitative Western blot analysis of platelet glycoproteins

Platelet lysates were prepared by lysing washed platelets in 50 mmol/L Tris (pH 6.8), 1% (vol/vol) Triton X-100, 10 mmol/L N-ethylmaleimide, 2 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 20 µmol/L leupeptin. After centrifugation at 1500g, the total protein concentration of the supernatants was measured by a bicinchoninic acid assay (Pierce, Rockford, IL). Comparable amounts of thrombasthenic and control total platelet protein were analyzed on an sodium dodecyl sulfate (SDS)-9% polyacrylamide gel under reducing conditions and transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon; Millipore, Bedford, MA). The membrane was incubated with a mixture of rabbit polyclonal antibodies directed against alpha IIb and beta 3 (both at a final concentration of 5 µg/mL), then detected with a 1:2000 dilution of alkaline phosphatase-conjugated goat-antirabbit IgG (Jackson Immunoresearch) and color development with the nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate substrate pair (Sigma, St Louis, MO).

Polymerase chain reaction amplification of alpha IIb and beta 3 fragments from genomic DNA

Genomic DNA was prepared from peripheral blood leukocytes and amplified by polymerase chain reaction (PCR) with Taq polymerase by using oligonucleotide primers from intronic flanking sequences of alpha IIb and beta 3. All exons of both genes were sequenced at least twice to exclude Taq errors. Exon 5 of beta 3 was amplified by using the sense primer 5'-CTCTACCAGTGACATGGCTGAA-3' and the antisense primer 5'-CAAGCTGAAACGAGCCCTGCC-3'. The PCR protocol entailed 5 minutes of denaturation at 100°C and 3 minutes of annealing at 56°C before the addition of Taq polymerase, then 30 cycles each of a 1-minute extension at 72°C, 45 seconds of denaturation at 96°C, and 45 seconds of annealing at 56°C. PCR products were extracted from agarose gels and analyzed by direct cycle sequencing. To detect multiple alleles from the patient LD and her parents, PCR products were subcloned into a TA overhang vector, pCRII (Invitrogen, Carlsbad, CA), and individual clones were sequenced.

PCR-based cartridge mutagenesis of beta 3 complementary DNA

Full-length complementary DNA (cDNA) encoding beta 3, mutated to delete an internal EcoRI cleavage site (a gift of Dr Gilbert C. White, University of North Carolina School of Medicine), was cloned into plasmid vector pBluescript SK (Stratagene, San Diego, CA). Overlap PCR was used to generate a fragment of beta 3 containing the T883C mutation found in patient LD. Briefly, primers with a single nucleotide mismatch at nucleotide 883 (sense primer, 5'-GGACGGAAGGCCGGCAGGCATTGTC-3', and antisense primer, 5'-GACAATGCCTGCCGGCCTTCCGTCC-3') were paired with wild-type beta 3 cDNA primers (sense primer, nucleotides 332-349: 5'-CCAGGTCACTCAAGTCAG-3'; and antisense primer, nucleotides 1799-1771:5'-GCAGGTGTCAGTAC-GCGTGGTACAGTTGC-3') to generate a 1467-base-pair (bp) cDNA fragment containing the T883C mutation. The mutated 332-1799 fragment was subcloned into pCRII and digested with the unique restriction enzymes NsiI and MluI, which produced a 966-bp mutated cartridge that was then ligated into NsiI- and MluI-digested wild-type beta 3-pBluescript SK. Finally, the reconstituted T883C beta 3 was excised from pBluescript SK with EcoRI and cloned into the mammalian expression vector pCDNA3 (Invitrogen). Sequence analysis of the T883C beta 3 construct was done to confirm the point mutation and proper insertion of the cartridge into the wild-type beta 3 cDNA.

Mammalian cell transfection

COS-7 cells were transfected with alpha IIb- and beta 3-containing expression vectors in the presence of diethylaminoethyl-dextran as described previously.30 After 48 to 60 hours of culture, transfected COS-7 cells were surface labeled with biotin or metabolically labeled with sulfur 35 (S35)-methionine, as described previously.30 The labeled cells were then solubilized in lysis buffer (50 mmol/L Tris-hydrochloric acid [HCl] at pH 7.4, 150 mmol/L NaCl, 1% (vol/vol) Triton X-100, and 2 mmol/L PMSF) and mixed for 30 minutes at 4°C. The lysates were centrifuged at 1600g for 30 minutes at 4°C and the supernatants stored at -80°C. For stable cell transfections, wild-type- and Leu262Probeta 3-pcDNA3 plasmids were introduced into human embryonal kidney 293 cells in the presence of cationic lipid (Lipofectamine; GIBCO BRL). Transfected cells were selected in media containing 0.7 mg/mL neomycin.

Pulse-chase metabolic labeling of COS-7 cells

Forty-eight hours after transfection, COS-7 cells were incubated for 30 minutes in Dulbecco modified Eagle medium (DMEM; Sigma) without methionine, then pulsed with 2.22 × 107 Bq/10-cm plate of 35S-methionine (DuPont-NEN, Boston, MA) for 30 minutes at 37°C. After the pulse, the cells were washed 3 times with Dulbecco phosphate-buffered saline (D-PBS) containing 1 mg/mL cold methionine (Sigma). The chase was begun by incubating the cells in DMEM containing 1 mg/mL cold methionine for 1, 4, 8, and 22 hours at 37°C. Labeled cells were washed 5 times in D-PBS containing 1 mg/mL cold methionine and then solubilized as described above.

Immunoprecipitation analysis

Lysates were precleared by incubating with 1% (wt/vol) BSA, 10 µg of preimmune mouse IgG (Sigma), and 50 µL of a 50% slurry of protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden) for 1 hour at room temperature with constant mixing. The beads were then separated by centrifugation at 1600g for 5 minutes, and the supernatant was incubated with 10 µg of mAb for 18 hours at 4°C, with mixing. Ten micrograms of rabbit antimouse IgG (DAKO, Carpinteria, CA) was added, and mixing was done for 1 hour at room temperature. Protein A-Sepharose (50 µL) was then added, and mixing continued for another hour at room temperature. The samples were then centrifuged at 1600g for 5 minutes and the supernatants discarded. The beads were washed 5 times in immunoprecipitation buffer (50 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, and 1% (vol/vol) Triton X-100), added to 50 µL of 2 × reducing buffer (4% SDS, 10% beta -mercaptoethanol, 100 mmol/L Tris-HCl [pH 6.8], 10% glycerol, and 0.001% bromophenol blue), and boiled for 10 minutes. Samples were centrifuged at 1600g for 5 minutes and the supernatants analyzed by SDS-polyacrylamide gel electrophoresis. Biotin-labeled samples were transferred to PVDF membrane and detected with horseradish peroxidase-streptavidin and chemiluminescence reagents as described previously.30 Gels containing 35S-methionine-labeled samples were fixed in 45% methanol and 12% acetic acid for 30 minutes, incubated in Enlightening solution (DuPont-NEN) for 30 minutes, dried, and analyzed by autoradiography.

Clot retraction by 293 cells expressing avbeta 3

Chromatographically purified fibrinogen I (gamma Agamma A) was a gift of Dr Michael W. Mosesson (Sinai-Samaritan Medical Center, Milwaukee, WI). Plasma from healthy subjects was depleted of fibronectin by passage through a gelatin-Sepharose column (Sigma) as described previously.24 Preliminary experiments (data not shown) demonstrated that untransfected 293 cells showed moderate retraction of clotted plasma but were unable to retract clots formed from fibronectin-depleted plasma. Therefore, fibronectin-depleted plasma was used in retraction assays to minimize the contribution of endogenous alpha vbeta 1 and other fibronectin receptors. Cultured untransfected and transfected 293 cells were washed twice in D-PBS (Sigma), lifted with 0.01% (wt/vol) trypsin and 3.5 mmol/L EDTA, and washed twice again in D-PBS. The cells (10 × 106/mL) were then resuspended in modified Eagle medium containing Earle salts (MEM-ES; Sigma) and 0.1 mmol/L L-glutamine. For clot-retraction assays, 300 µL of the cell solution was added to 100 µL of medium containing 5 µg aprotinin (Sigma) and either 100 µL of fibronectin-depleted plasma or 100 µL of medium containing 100 µg of purified fibrinogen, in a 10- × 75-mm borosilicate glass tube (Fisher Scientific, Pittsburgh, PA). To initiate clot formation, 1 U of human alpha  thrombin was added and the tubes were incubated at 37°C. The external dimensions of each clot were measured at 30-minute intervals, and a cylindrical model (volume = pi radius2 × height) was used to calculate the approximate clot volume.

In some experiments, plasma or 293 cells were preincubated with peptides or mAbs for 15 minutes at 22°C. The peptides RGDW and RGEW and the fibrinogen peptide HHLGGAKQAGDV (H12), corresponding to gamma -chain residues 400 to 411, were synthesized on a Pepsynthesizer (Model 9050; Millipore) with 9-fluoronylmethoxycarbonyl chemistry using standard manufacturers' procedures.

Cell adhesion to immobilized substrates

Adhesion of fluorescently labeled cells was preformed as described previously.8 Protein substrates or antibodies (2-10 µg/mL in D-PBS) were added to an Immulon 2 96-well plate (Dynatech, Chantilly, VA) and incubated overnight at 4°C. Before use, the coated plates were washed with D-PBS and blocked with 1% (wt/vol) BSA in D-PBS for 1 hour at 22°C. Transfected or control 293 cells were washed and trypsinized as described above, resuspended at 2 × 106 cells/mL in MEM-ES medium, and incubated with 2 µg/mL calcein AM (Molecular Probes, Eugene, OR) for 30 minutes at 37°C. After labeling, the cells were washed twice in D-PBS and resuspended in MEM-ES at 3 × 106/mL. One hundred microliters of labeled cell suspension was added to each well and allowed to adhere for 1 hour at 37°C. The total fluorescence per well was measured in a multiwell plate reader (Cytofluor; Perseptive Biosystems, Framingham, MA) immediately after incubation and 2 washes with MEM-ES to remove nonadherent cells.


    Results
Top
Abstract
Introduction
Patients and methods
Results
Discussion
Acknowledgments
References

The clinical history of patient LD indicated a marked hemostatic defect. Platelets from the patient did not aggregate in response to adenosine diphosphate, epinephrine, or arachidonic acid and showed only shape change in response to collagen, but they agglutinated with ristocetin (Figure 1). The failure of the platelets to support fibrinogen-dependent aggregation suggests a defect in platelet alpha IIbbeta 3.


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Fig 1. Platelets from the proband (LD) do not support fibrinogen-dependent platelet aggregation. Platelet-rich plasma (PRP) from patient LD was stirred in an aggregometer at 37°C before the addition of agonists (time point indicated by arrow). Platelets from LD showed no aggregation in response to adenosine diphosphate (ADP), epinephrine (Epi), or arachidonic acid (AA) and showed shape change only in response to collagen (Coll). Platelets from LD agglutinated reversibly with high-dose ristocetin.

On flow cytometry (Figure 2A), platelets from LD showed reduced (30% of normal) binding of subunit-specific mAbs against alpha IIb or beta 3 but no binding of a complex-specific antibody (AP2). Total platelet levels of alpha IIbbeta 3 were assessed by semiquantitative immunoblotting of platelet lysates (Figure 2B). In lysates from LD, faint but detectable bands of the expected apparent molecular weight were seen, corresponding to approximately 10% of control levels. These findings are consistent with a diagnosis of Type II GT. In both assays, platelets from the proband's father showed reduced (50% of normal) levels of platelet alpha IIbbeta 3, findings consistent with a heterozygous defect that affects alpha IIbbeta 3 expression. No platelet samples from the patient's mother were available. The failure of platelets from LD to aggregate implies a defect in binding of soluble fibrinogen.


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Fig 2. Platelets from LD express alpha IIb and beta 3 subunits but little alpha IIbbeta 3 complex. (A) Flow-cytometric analysis of surface alpha IIbbeta 3. Monoclonal antibodies directed against a control glycoprotein, GPIb (AP1), the alpha IIbbeta 3 complex (AP2), the beta 3 subunit (AP3), or the alpha IIb subunit (TAB) were incubated with washed platelets from a healthy donor (control), the patient (LD), or the patient's father (F), and binding was assessed. Control platelet binding of nonimmune mouse IgG yielded a relative mean fluorescence of 2. (B) Western blot analysis of total platelet alpha IIbbeta 3. The indicated amounts of Triton X-100-treated platelet lysates from a healthy control, the patient (LD), and the patient's father (F) were separated by sodium dodecyl sulfate (SDS)-7% polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and detected with rabbit polyclonal antibodies against alpha IIb and beta 3. The positions of alpha IIb (upper arrow) and beta 3 (lower arrow) are indicated.

The ability of platelets from LD to interact with fibrin was assessed in a clot-retraction assay. Clotted PRP from LD (Figure 3) and both parents (data not shown) retracted as strongly as control plasma, indicating that the defective platelet alpha IIbbeta 3 complex was still capable of interacting with fibrin.


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Fig 3. Platelets from LD retract a fibrin clot. PRP from a healthy control or patient LD was diluted with autologous plasma to the indicated platelet concentration and clotted by adding 8 mmol/L calcium chloride. After 1 hour of incubation at 37°C, clot retraction of platelets from LD was equivalent to that of control platelets.

Genetic analysis of LD and her family (Figure 4) demonstrated that LD was a compound heterozygote for 2 novel mutations of the beta 3 gene in exon 5. First, in both LD and her father, a dinucleotide deletion of bases GC867 to GC868 was found. This deletion is predicted to result in a frameshift and substitution of a premature stop codon for amino acid 267. Second, LD and her mother had a point mutation of T883C, which is predicted to cause the substitution of a Pro for Leu at position 262. Sequencing of both alleles by subcloning of PCR-amplified genomic DNA confirmed that each of LD's parents carried 1 wild-type and 1 mutant beta 3 allele, whereas LD had inherited both exon 5 mutations. No mutations were found in the alpha IIb gene of LD. The half-normal levels of alpha IIbbeta 3 observed on platelets from LD's father (Figure 2) are consistent with failure to express the truncated D867-868 form of beta 3. This suggests that all the beta 3 present on platelets from LD carries the Leu262Pro mutation.


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Fig 4. LD is a compound heterozygote for 2 mutations of the beta 3 gene. Analysis of genomic DNA amplified by polymerase chain reaction identified a deletion of GC867-868 in LD's father (F) and a T883C substitution in her mother (M). Both mutant alleles were found in LD. The patient's siblings were not analyzed but are asymptomatic.

Comparison of the human beta 3 amino acid sequence with beta 3 subunits from other species and other human beta  integrin subunits showed that the position corresponding to beta 3 262 was always occupied by a Leu residue (Figure 5A). The effect of a Leu262-to-Pro substitution on the predicted structure of beta 3 residues 200 to 300 was modeled by using a selection of protein structure algorithms (GeneWorks; Intelligenetics, Mountain View, CA). Compared with the wild type, the presence of a Pro at position 262 led to a reduction in hydrophobicity (by the Kyle-Doolittle hydrophobicity-scoring system), a reduced probability of an alpha  helix but an increased probability of a beta  turn (by Garnier protein-structure analysis), and an increased probability of surface exposure (Figure 5B). Therefore, it is likely that the Pro262beta 3 mutant adopts a local conformation different from that of Leu262beta 3.


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Fig 5. Structural analysis of the Leu262Probeta 3 mutation. (A) Leucine (Leu) 262 is completely conserved among beta  subunits. The amino acid sequence of human beta 3 residues cystine (Cys) 232 to Cys273 is shown aligned with frog (Xenopus) and mouse beta 3 and with the sequences of 7 other human beta  subunits. Notably, the Leu residue at position 262 (in boldface) is invariant. The proposed intramolecular disulfide loop Cys232-27330 is shown. (B) Predicted hydrophobicity and secondary structure are shown for beta 3 amino acids 200 to 300 containing either wild-type Leu262 (black line) or mutant Pro262 (stippled line with arrow). The position of amino acid 262 is indicated by the vertical line.

Mammalian cell-expression systems were used to confirm that the Leu262Pro form of alpha IIbbeta 3 showed altered function. Leu262Probeta 3 cDNA was constructed and cotransfected with wild-type alpha IIb into COS-7 cells. Biotin surface labeling of these transiently transfected cells showed that the mutant complex was poorly expressed and less stable than the wild type: from a detergent lysate, the complex-specific antibody AP2 immunoprecipitated only trace amounts of alpha IIbLeu262Probeta 3 (data not shown). The beta 3 subunit-specific antibody, AP3, precipitated both subunits from lysates of wild-type alpha IIbbeta 3 transfected cells but only the beta 3 subunit from cells transfected with alpha IIbLeu262Probeta 3 (data not shown), resultsconsistent with the idea that the alpha IIbLeu262Probeta 3 complex is easily dissociable. The addition of 2 mmol/L calcium to alpha IIbLeu262Probeta 3 lysates did not preserve subunit association. Similarly, the alpha IIb-specific antibody, Tab, captured only the alpha IIb subunit from lysates of cells transfected with the mutant receptor (data not shown). Thus, the surface expression of alpha IIbLeu262Probeta 3 in COS-7 cells recapitulated the platelet phenotype, with both subunits detectable but little or no binding of a complex-specific antibody.

Metabolic labeling of transfected COS-7 cells with 35S-methionine was used to assess the biosynthesis of alpha IIbLeu262Probeta 3. Normal synthesis and expression of alpha IIbbeta 3 follows an ordered series of events31,32 in which cleavage of prealpha IIb to mature alpha IIb indicates that the nascent integrin complex has reached the Golgi and undergone posttranslational processing. Pulse-chase experiments were used to follow the intracellular maturation of alpha IIb and either wild-type beta 3 or Leu262Probeta 3. As shown in Figure 6, immunoprecipitation of Leu262Probeta 3 by an anti-beta 3 antibody captured prealpha IIb, indicating that the mutant subunit had formed a heterodimer in the endoplasmic reticulum (ER). There was no apparent defect in the extent or rate of Leu262Probeta 3 synthesis. However, the maturation of the mutant complex was markedly delayed. In cells expressing alpha IIbbeta 3, mature alpha IIb was observed within 4 hours of the methionine pulse and cleavage was virtually complete by 22 hours. In contrast, prealpha IIb associated with Leu262Probeta 3 was not cleaved before 22 hours, a finding consistent with a defect in heterodimer export from the ER. These results provide a likely molecular explanation for the reduced expression of alpha IIbLeu262Probeta 3 observed in platelets from LD.


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Fig 6. Maturation of alpha IIbLeu262Probeta 3 is delayed in COS-7 cells. COS-7 cells transfected with wild-type (WT) alpha IIb and either WT beta 3 or Leu262Probeta 3 (LD) were pulsed with sulfur 35-methionine for 30 minutes and chased for up to 22 hours with medium containing 1 mg/mL cold methionine. Cell lysates prepared at the indicated times were immunoprecipitated with an anti-beta 3 antibody (AP3) and analyzed by autoradiography of an SDS-7% polyacrylamide gel run under reducing conditions. Although prealpha IIb and Leu262Probeta 3 synthesis and association are normal in lysates from LD, the appearance of a mature alpha IIbLeu262Probeta 3 complex is markedly delayed.

To study the effect of the Leu262Probeta 3 on ligand binding and specificity, the mutant beta 3 subunit alone was stably expressed in human embryonal kidney 293 cells. Previous studies using this cell line23 showed that transfected beta 3 is expressed on the cell surface in a complex with endogenous alpha v and can mediate fibrin-clot retraction. A further advantage of this system is that alpha vbeta 3, unlike transfected alpha IIbbeta 3, does not require activation to bind ligand.24 The expression of alpha v-Leu262Probeta 3 in 293 cells was assessed by flow cytometry and biotin surface labeling. As shown in Figure 7, the mutant complex was expressed less effectively than wild-type alpha vbeta 3 (40%-50% levels), as expected, but intact heterodimer was detected on the cell surface by both AP3 and the complex-specific antibody, LM609.


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Fig 7. Leu262Probeta 3 is expressed in a complex with alpha v in 293 cells. Untransfected (C) 293 cells or cells stably transfected with WT beta 3 or Leu262Probeta 3 (LD) were analyzed by flow cytometry. The mean fluorescence for each cell line is shown. The 2 left-hand panels show binding of a negative-control antibody (nonimmune mouse IgG) and a positive-control antibody (polyclonal anti-beta 1). The 2 right-hand panels show the results of flow cytometry and immunoprecipitation of biotin surface-labeled cells with antibodies against beta 3 (AP3) or the alpha vbeta 3 complex (LM609). The positions of alpha v and beta 3 are indicated.

The ability of alpha vLeu262Probeta 3 cells to retract a fibrin clot was compared with that of untransfected and alpha vbeta 3 cells (Figure 8). Wild-type alpha vbeta 3 cells mediated rapid retraction of fibrin clots from both fibronectin-depleted plasma (Figure 8A) and purified fibrinogen (Figure 8B). Despite the reduced receptor density on their surface, alpha vLeu262Probeta 3 cells showed nearly normal retraction of fibrin clots (Figure 8). In both alpha vbeta 3 and alpha vLeu262Probeta 3 cells, retraction could be enhanced by 0.5-mmol/L Mn2+ and inhibited by EDTA or ethylene glycol tetraacetic acid (data not shown), inhibited by RGDW but not by RGEW or fibrinogen gamma -chain peptides, and inhibited by the complex-specific antibody, LM609 (Figure 8C). This suggests that a similar, cation-dependent mechanism underlies the interaction of both the wild-type and mutant integrin complex with an RGD-like site on fibrin.


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Fig 8. alpha vLeu262Probeta 3 on 293 cells retracts a fibrin clot. Untransfected 293 cells (solid circles) and cells expressing WT alpha vbeta 3 (solid squares) or alpha vLeu262Probeta 3 (open circles) were assessed for their ability to retract clotted fibronectin-depleted plasma (A) or purified fibrinogen (B). The estimated clot volume is expressed as a function of time (minutes). The data shown are representative results from 3 separate experiments. alpha vLeu262Probeta 3-mediated clot retraction was comparable to that of WT alpha vbeta 3. (C) WT alpha vbeta 3 cells (open columns) or alpha vLeu262Probeta 3 cells (solid columns) were incubated for 15 minutes at 22°C with peptides RGDW, RGEW, or H12 (all at a final concentration of 2 mmol/L) or with the indicated antibodies (final concentration, 10 µg/mL). Only RGDW and the alpha vbeta 3-specific antibody LM609 fully inhibited clot retraction.

Finally, transfected 293 cells were assessed for their ability to bind purified fibrinogen and other ligands immobilized in a 96-well microtiter plate (Figure 9). Untransfected cells and beta 3-transfected lines bound equally to the control ligand fibronectin, suggesting a primary role for alpha vbeta 1 and other endogenous integrins. The degree of cell spreading on fibronectin after adhesion was similar for all cell lines. Similarly, all 293 cell lines adhered to immobilized vitronectin (data not shown). As expected, both beta 3-transfected cell lines, but not untransfected cells, bound to a beta 3-specific antibody, AP3. However, whereas wild-type alpha vbeta 3 cells showed significant adhesion and spreading on immobilized fibrinogen, the alpha vLeu262Probeta 3 complex was unable to interact with immobilized fibrinogen. Therefore, the expression of a alpha vLeu262Probeta 3 mutation in a cultured cell line recapitulated the ligand-binding specificity of alpha IIbLeu262Probeta 3 on platelets in that it was unable to bind fibrinogen but showed nearly normal binding and retraction of a fibrin gel.


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Fig 9. alpha vLeu262Probeta 3 does not support cell adhesion to immobilized fibrinogen. Untransfected 293 cells (open columns) and cells expressing WT alpha vbeta 3 (hatched columns) or alpha vLeu262Probeta 3 (solid columns) were labeled with calcein AM and allowed to adhere to immobilized substrates (bovine serum albumin, AP3, fibronectin, or fibrinogen) for 1 hour at 37°C. Adhesion is shown as the ratio of fluorescence per well before and after washing. Each column represents the mean (± SD) value from triplicate samples, and the results are representative of 4 separate experiments.


    Discussion
Top
Abstract
Introduction
Patients and methods
Results
Discussion
Acknowledgments
References

In this study, we characterized the molecular defects responsible for a novel case of GT. The patient's history and the absence of platelet aggregation in response to multiple agonists allowed the clinical diagnosis of GT. On the basis of our detection by flow cytometry and immunoblotting of platelet alpha IIbbeta 3 levels that were 5% to 30% of normal values, we found this patient to have type II thrombasthenia. Although flow data with subunit-specific mAbs showed surface expression to be 30% of normal levels, there was no binding of the complex-specific mAb, AP2 (Figure 2A). This indicates that either the conformation of the mutant complex is abnormal or that the complex itself is unstable after surface expression. Such an unstable complex was described previously in another beta 3 point mutation (serine 162 to Leu), in which dissociation of the mutant alpha IIbbeta 3 complex was demonstrated by abnormal migration through a sucrose gradient.33

Genotypic analysis of the patient's family identified 2 novel mutations of beta 3 only 16 bases apart in exon 5. The first was a dinucleotide deletion of bases 867 to 868 that resulted in a frameshift and substitution of a termination codon for amino acid residue 267. The 867-868 deletion was found in both the patient and her father and is predicted to result in the synthesis of a severely truncated form of beta 3 encoding only residues 1 to 267. The father's phenotype, with levels of platelet alpha IIbbeta 3 that were 50% of normal values, suggests that this truncation mutant interferes significantly with receptor expression. Several truncation mutants affecting the beta 3 subunits were previously described in patients with GT.34-36 In all these cases, the truncated subunit failed to be expressed, thereby conferring a type I GT phenotype. In vitro studies with truncated recombinant alpha IIb and beta 3 molecules showed that beta 3 fragments corresponding to residues 111 to 31814 alone are sufficient for heterodimer formation with wild-type or truncated forms of alpha IIb. However, it appears that heterodimer formation is not sufficient for surface expression of the receptor; transfection studies with alpha IIb-truncation mutants37,38 found that mutant heterodimers were retained in the ER and degraded. By analogy with these mutants, it is likely that the truncated 1-267 form of beta 3 is also subject to intracellular trapping and is not expressed on the cell surface.

The second mutation found in this family resulted in the substitution of a Pro for Leu at residue 262. Because the 867-868-deletion mutant allele is unlikely to be expressed, all the beta 3 detectable on platelets from LD can be expected to contain the Leu262Pro mutation. In COS cells, cotransfection of wild-type alpha IIb and Leu262Probeta 3 reproduced the platelet phenotype, with reduced levels of subunit expression on the cell surface and a heterodimer complex that was unstable in detergent lysates and not recognized by the complex-specific antibody, AP2. Analysis of the intracellular processing of alpha IIbLeu262Probeta 3 (Figure 6) showed that maturation of the mutant complex was markedly delayed. Delayed intracellular trafficking was observed in other cases of type II thrombasthenia due to point mutations of alpha IIb31,37 and beta 3,33 suggesting that an altered heterodimer conformation interferes with receptor processing. The presence of detectable surface expression of Leu262Probeta 3 in both platelets from LD (Figure 2) and COS cells shows that some mutant alpha IIbLeu262Probeta 3 receptor can still undergo posttranslational processing and be exported from the Golgi complex. Therefore, the reduced levels of receptor on platelets from LD may reflect both delayed intracellular trafficking and reduced stability of the heterodimer in the plasma membrane.

The alpha IIbLeu262Probeta 3 complex on platelets from LD was unable to support fibrinogen-dependent platelet aggregation (Figure 1) but could bind fibrin (Figure 3) in a clot-retraction assay. We studied the ligand-binding specificity of Leu262Probeta 3 by expressing it in a complex with endogenous alpha v subunit in human embryonal kidney 293 cells. Because 293 cells express the vitronectin receptor alpha vbeta 1 but have no endogenous beta 3,39 transfection of beta 3 into these cells enables them to mediate fibrin-clot retraction.24 The alpha vLeu262Probeta 3 complex expressed in stably transfected 293 cells had the same ligand specificity as alpha IIbLeu262Probeta 3: it bound and retracted fibrin (Figure 8) but did not adhere to immobilized fibrinogen (Figure 9). The small reduction in clot retraction observed in cells expressing alpha vLeu262Probeta 3 was likely due to quantitative differences in surface-expression levels or qualitative differences in the receptor-ligand interaction. Clot retraction mediated by alpha vbeta 3 and alpha vLeu262Probeta 3 appeared to proceed by means of the same mechanism; both were cation dependent, enhanced in the presence of Mn2+, and inhibited by EDTA.

Consistent with results in earlier studies in platelets20,40 and nucleated cells,24 an RGD-containing peptide inhibited fibrin-clot retraction by both cell types. Despite this, fibrin RGD sequences may not be required for integrin binding; earlier studies indicated that the RGD at Aalpha 95-97 was not involved in alpha IIbbeta 3 binding to fibrinogen,41 and fibrinogen lacking the RGD motif at Aalpha 572-574 still supported alpha vbeta 3-dependent clot retraction.42 We did not observe any inhibition by a fibrinogen gamma A 400-412 peptide, in keeping with reports that the gamma A region is also not required for alpha IIbbeta 3-mediated fibrin binding.13,41 These studies of mutant ligand have raised the possibility that fibrinogen and fibrin bind to different or overlapping sites on alpha IIbbeta 3.12,13 Our description of a mutant receptor that differentiates between binding fibrin and fibrinogen is further evidence for ligand-specific molecular interactions. The preservation of fibrin binding in the Leu262Pro mutant suggests that receptor conformation or stability (or both) is less critical for binding to multimeric fibrin than to fibrinogen.

The Leu at position 262 in beta 3 was highly conserved among beta  integrins (Figure 5), suggesting that it may play a role in maintaining receptor structure. Leu262 lies within a proposed intramolecular disulfide loop, between Cys232 and Cys273.30 Limited tryptic proteolysis of alpha IIbbeta 3 and N-terminal sequencing of the resulting fragments found beta 3 residues 217 to 298 associated with alpha IIbH residues 91-139 and alpha IIbL residues 1 to 26,43 suggesting that this region of beta 3 is involved in intersubunit contacts. Therefore, the Leu262Pro substitution may affect complex conformation and stability by disrupting a beta 3 contact site for alpha IIb. A mAb that recognizes beta 3 residues 262 to 302, P40, was also characterized.44 P40 was reported to bind platelet alpha IIbbeta 3 only after treatment with EDTA, whereas it could bind endothelial cell alpha vbeta 3 without receptor dissociation.44 This implies, first, that in alpha IIbbeta 3, the beta 3 262-302 epitope is not surface exposed, consistent with the hypothesis that it forms part of an interface with alpha IIb, and second, that the conformation of beta 3 in alpha IIbbeta 3 differs from that of beta 3 complexed to alpha v. A comparison of the poor expression levels of alpha IIbLeu262Probeta 3 in both platelets and COS cells with the high expression of alpha vLeu262Probeta 3 in 293 cells indeed indicates that the conformation of the 232-273 loop is more critical for association with alpha IIb than with alpha v.

Sequences immediately adjacent to the 232-273 loop of beta 3 have been implicated in ligand binding. Several research groups have observed that beta 3 peptides corresponding to residues 211 to 231 can inhibit fibrinogen binding to alpha IIbbeta 3,17,45,46 but they disagreed about whether the peptide bound fibrinogen17,45 or interacted with alpha IIbbeta 3 itself.46 One study reconciled these viewpoints by reporting that the beta 3 peptide 214-218 binds alpha IIbbeta 3, whereas a 217-231 peptide binds fibrinogen.47 In addition, structural modeling and mutagenesis of beta 3 identified both Asp217 and glutamic acid 220 as potential cation-coordinating residues in the MIDAS-like domain of beta 3.7,48 On the C-terminal side, a recombinant fragment comprising beta 3 residues 274 to 368 was found to bind to the gamma  chain of fibrinogen and a mAb against residues 274 to 403 inhibited fibrinogen binding to platelets, suggesting that this region of beta 3 includes a fibrinogen-binding site.49 Therefore, the intrachain disulfide loop containing Leu262 may not only contribute to heterodimer formation but may also be essential for the orientation of flanking binding sites.

Most studies of alpha IIbbeta 3-ligand interactions focused on the cation-dependent binding of soluble fibrinogen and the undefined process of receptor activation that must precede binding. Platelet adhesion to immobilized fibrinogen can proceed without alpha IIbbeta 3 activation, possibly because of the exposure of binding sites on fibrinogen after surface adhesion.50 However, a fibrin clot presents a third set of conditions to alpha IIbbeta 3 in that (1) activation of the complex is required for efficient binding,20,25 (2) the molecular structure of cross-linked fibrin differs from that of monomeric fibrinogen,51 and (3) the receptor recognizes motifs other than the gamma -chain C-terminal.12,13 The in vitro phenomenon of clot retraction and the ability of certain thrombasthenic alpha IIbbeta 3 mutants to retain fibrin binding provide a powerful approach for exploring the alpha IIbbeta 3-fibrin interaction. Additional selective mutagenesis studies of beta 3 residues 232 to 273 based on the above findings may clarify the regulatory role of this intrachain loop in receptor activation.


    Acknowledgments
Top
Abstract
Introduction
Patients and methods
Results
Discussion
Acknowledgments
References

We thank Sabine Weyerbusch-Bottum for technical assistance, Dr Ronggang Wang and Dr Michael Mosesson for valuable discussions, Trudy Holyst for custom peptide synthesis, and the molecular biology core facility of the Blood Research Institute for automated DNA sequencing.


    Footnotes

Submitted October 9, 1997; accepted February 23, 2000.

Supported by grant P01-HL-44612 from the National Institutes of Health and performed during the tenure of an Established Investigator Award from the American Heart Association (P.J.N.). C.M.W. was the recipient of a Winthrop Traveling Fellowship from the Royal Australasian College of Physicians and was a postdoctoral fellow of the Wisconsin Affiliate of the American Heart Association (96-F-Post-49).

Reprints: Peter J. Newman, Blood Research Institute, The Blood Center of Southeastern Wisconsin, PO Box 2178, Milwaukee, 53201-2178; e-mail: pjn{at}smtpgate.bcsew.edu.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.


    References
Top
Abstract
Introduction
Patients and methods
Results
Discussion
Acknowledgments
References

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