A Leu262Pro mutation in the integrin β₃ subunit results in an α_IIb-β₃ complex that binds fibrin but not fibrinogen

The aggregation of platelets and subsequent thrombus formation is mediated by the platelet-specific integrin α_IIbβ₃ (glycoprotein IIb-IIIa). Platelet α_IIbβ₃ is a heterodimeric surface receptor consisting of an alpha (α_IIb) and a beta (β₃) subunit in a noncovalent, cation-dependent complex.1 Platelet aggregation depends on α_IIbβ₃ binding of fibrinogen,2but the receptor can recognize other ligands, including fibronectin, von Willebrand factor (vWf), and vitronectin.3α_IIbβ₃ 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 α_IIbβ₃ structure and function is the rare, autosomal recessive bleeding disorder Glanzmann thrombasthenia (GT), in which α_IIbβ₃ is dysfunctional or absent.4 

GT mutations characterized at the molecular level are heterogenous and affect the α_IIb and β₃ genes equally.4,5 In many patients, gene deletions, frameshifts, and premature terminations result in α_IIbβ₃expression at less than 5% of normal levels (type I GT). Of more interest are cases in which a dysfunctional α_IIbβ₃ 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 β₃ gene in 2 critical areas. First, mutations of the charged residues arginine (Arg) 119, Arg214, and Arg216⁴ in the β₃ 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 α subunits.6,7 Second, mutations in the cytoplasmic domain of β₃, 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 α_IIbβ₃ that participate in ligand binding and receptor activation.

The molecular events involved in α_IIbβ₃ligand binding have been partly described; like many integrins, α_IIbβ₃ 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 Aα, Bβ, and γ chains, and contains 4 RGD motifs, 2 in each Aα 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 γ chain, binds α_IIbβ₃ 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 Aα-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 α_IIb (residues 1-233) and β₃ (residues 111-318) formed an RGD-binding complex.14 Cross-linking and peptide studies identified β₃ 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 β₃. In α_IIb, the γ-chain peptide of fibrinogen was cross-linked to residues 294 to 314 of α_IIb, suggesting that this is also a ligand-recognition site,18 and mutation of α_IIb residues Gly184 to Gly193 was reported to abolish fibrinogen binding.19 

The molecular interactions of α_IIbβ₃ 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 β₃ subunit is important in binding fibrin, triggering retraction through links between the cytoplasmic domain of β₃ and cytoskeletal proteins.23 In cultured mammalian cell lines, β₃, as part of the vitronectin receptor, α_vβ₃, induced spontaneous fibrin-clot retraction, whereas α_IIbβ₃ required prior activation of the integrin for retraction.24,25 In contrast to the situation with platelet aggregation, the C-terminal of the fibrinogen γ chain does not appear to be required for fibrin-clot retraction mediated by α_IIbβ₃,12 13 suggesting that fibrinogen and fibrin may bind to different sites on the receptor.

In the current study, we identified a novel β₃ 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 β₃located between 2 putative ligand-binding sites and involved in subunit interactions. By expressing Leu262Proβ₃ in complex with α_v in mammalian cells, we demonstrated that the mutant receptor binds fibrin in a similar manner to wild-type β₃but does not recognize fibrinogen.

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.

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.

The α_IIb-specific monoclonal antibody (mAb) Tab was a gift from Dr Rodger McEver (University of Oklahoma).26 The β₃-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 α_IIbβ₃,28 were provided by Dr Robert R. Montgomery (Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, WI). The mAb LM609, which recognizes the α_vβ₃ complex,29 was a gift from Dr David A. Cheresh (Scripps Institute, La Jolla, CA). The mAb P4C10 against human β1 integrin was obtained from GIBCO BRL (Gaithersburg, MD). Rabbit polyclonal anti-α_IIb and anti-β₃ antibodies were prepared at the Blood Research Institute by using standard procedures.

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 × 10⁷platelets 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).

Platelet lysates were prepared by lysing washed platelets in 50 mmol/L Tris (pH 6.8), 1% (vol/vol) Triton X-100, 10 mmol/LN-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 α_IIb and β₃ (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).

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 α_IIb and β₃. All exons of both genes were sequenced at least twice to exclude Taq errors. Exon 5 of β₃ 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.

Full-length complementary DNA (cDNA) encoding β₃, 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 β₃containing 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 β₃ 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 intoNsiI- and MluI-digested wild-type β₃–pBluescript SK. Finally, the reconstituted T883C β₃ was excised from pBluescript SK with EcoRI and cloned into the mammalian expression vector pCDNA₃(Invitrogen). Sequence analysis of the T883C β₃ construct was done to confirm the point mutation and proper insertion of the cartridge into the wild-type β₃ cDNA.

COS-7 cells were transfected with α_IIb- and β₃-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.30The 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 Leu262Proβ₃-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.

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 × 10⁷Bq/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.

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% β-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 containing³⁵S-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.

Chromatographically purified fibrinogen I (γAγA) 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 α_vβ1 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/LL-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 α 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 = πradius2 × 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 γ-chain residues 400 to 411, were synthesized on a Pepsynthesizer (Model 9050; Millipore) with 9-fluoronylmethoxycarbonyl chemistry using standard manufacturers' procedures.

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.

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 α_IIbβ₃.

On flow cytometry (Figure 2A), platelets from LD showed reduced (30% of normal) binding of subunit-specific mAbs against α_IIb or β₃ but no binding of a complex-specific antibody (AP2). Total platelet levels of α_IIbβ₃ 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 α_IIbβ₃, findings consistent with a heterozygous defect that affects α_IIbβ₃ 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.

The ability of platelets from LD to interact with fibrin was assessed in a clot-retraction assay. Clotted PRP from LD (Figure3) and both parents (data not shown) retracted as strongly as control plasma, indicating that the defective platelet α_IIbβ₃ complex was still capable of interacting with fibrin.

Genetic analysis of LD and her family (Figure4) demonstrated that LD was a compound heterozygote for 2 novel mutations of the β₃ 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 β₃ allele, whereas LD had inherited both exon 5 mutations. No mutations were found in the α_IIb gene of LD. The half-normal levels of α_IIbβ₃observed on platelets from LD's father (Figure 2) are consistent with failure to express the truncated D867-868 form of β₃. This suggests that all the β₃ present on platelets from LD carries the Leu262Pro mutation.

Comparison of the human β₃ amino acid sequence with β₃ subunits from other species and other human β integrin subunits showed that the position corresponding to β₃ 262 was always occupied by a Leu residue (Figure5A). The effect of a Leu262-to-Pro substitution on the predicted structure of β₃ 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 α helix but an increased probability of a β turn (by Garnier protein-structure analysis), and an increased probability of surface exposure (Figure 5B). Therefore, it is likely that the Pro262β₃ mutant adopts a local conformation different from that of Leu262β₃.

Mammalian cell-expression systems were used to confirm that the Leu262Pro form of α_IIbβ₃ showed altered function. Leu262Proβ₃ cDNA was constructed and cotransfected with wild-type α_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 α_IIbLeu262Proβ₃ (data not shown). The β₃ subunit-specific antibody, AP3, precipitated both subunits from lysates of wild-type α_IIbβ₃transfected cells but only the β₃ subunit from cells transfected with α_IIbLeu262Proβ₃ (data not shown), resultsconsistent with the idea that the α_IIbLeu262Proβ₃ complex is easily dissociable. The addition of 2 mmol/L calcium to α_IIbLeu262Proβ₃ lysates did not preserve subunit association. Similarly, the α_IIb-specific antibody, Tab, captured only the α_IIb subunit from lysates of cells transfected with the mutant receptor (data not shown). Thus, the surface expression of α_IIbLeu262Proβ₃ 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 with³⁵S-methionine was used to assess the biosynthesis of α_IIbLeu262Proβ₃. Normal synthesis and expression of α_IIbβ₃ follows an ordered series of events31 32 in which cleavage of preα_IIb to mature α_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 α_IIb and either wild-type β₃ or Leu262Proβ₃. As shown in Figure 6, immunoprecipitation of Leu262Proβ₃ by an anti-β₃ antibody captured preα_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 Leu262Proβ₃synthesis. However, the maturation of the mutant complex was markedly delayed. In cells expressing α_IIbβ₃, mature α_IIb was observed within 4 hours of the methionine pulse and cleavage was virtually complete by 22 hours. In contrast, preα_IIb associated with Leu262Proβ₃ 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 α_IIbLeu262Proβ₃ observed in platelets from LD.

To study the effect of the Leu262Proβ₃ on ligand binding and specificity, the mutant β₃ subunit alone was stably expressed in human embryonal kidney 293 cells. Previous studies using this cell line23 showed that transfected β₃is expressed on the cell surface in a complex with endogenous α_v and can mediate fibrin-clot retraction. A further advantage of this system is that α_vβ₃, unlike transfected α_IIbβ₃, does not require activation to bind ligand.24 The expression of α_v-Leu262Proβ₃ in 293 cells was assessed by flow cytometry and biotin surface labeling. As shown in Figure7, the mutant complex was expressed less effectively than wild-type α_vβ₃ (40%-50% levels), as expected, but intact heterodimer was detected on the cell surface by both AP3 and the complex-specific antibody, LM609.

The ability of α_vLeu262Proβ₃ cells to retract a fibrin clot was compared with that of untransfected and α_vβ₃ cells (Figure8). Wild-type α_vβ₃ 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, α_vLeu262Proβ₃ cells showed nearly normal retraction of fibrin clots (Figure 8). In both α_vβ₃ and α_vLeu262Proβ₃ 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 γ-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.

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 β₃-transfected lines bound equally to the control ligand fibronectin, suggesting a primary role for α_vβ1 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 β₃-transfected cell lines, but not untransfected cells, bound to a β₃-specific antibody, AP3. However, whereas wild-type α_vβ₃ cells showed significant adhesion and spreading on immobilized fibrinogen, the α_vLeu262Proβ₃ complex was unable to interact with immobilized fibrinogen. Therefore, the expression of a α_vLeu262Proβ₃ mutation in a cultured cell line recapitulated the ligand-binding specificity of α_IIbLeu262Proβ₃ on platelets in that it was unable to bind fibrinogen but showed nearly normal binding and retraction of a fibrin gel.

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 α_IIbβ₃levels 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 β₃ point mutation (serine 162 to Leu), in which dissociation of the mutant α_IIbβ₃complex was demonstrated by abnormal migration through a sucrose gradient.33 

Genotypic analysis of the patient's family identified 2 novel mutations of β₃ 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 β₃ encoding only residues 1 to 267. The father's phenotype, with levels of platelet α_IIbβ₃ that were 50% of normal values, suggests that this truncation mutant interferes significantly with receptor expression. Several truncation mutants affecting the β₃ 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 α_IIb and β₃ molecules showed that β₃ fragments corresponding to residues 111 to 31814 alone are sufficient for heterodimer formation with wild-type or truncated forms of α_IIb. However, it appears that heterodimer formation is not sufficient for surface expression of the receptor; transfection studies with α_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 β₃ 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 β₃ detectable on platelets from LD can be expected to contain the Leu262Pro mutation. In COS cells, cotransfection of wild-type α_IIb and Leu262Proβ₃ 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 α_IIbLeu262Proβ₃(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 α_IIb31,37 and β₃,33suggesting that an altered heterodimer conformation interferes with receptor processing. The presence of detectable surface expression of Leu262Proβ₃ in both platelets from LD (Figure 2) and COS cells shows that some mutant α_IIbLeu262Proβ₃ 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 α_IIbLeu262Proβ₃ 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 Leu262Proβ₃ by expressing it in a complex with endogenous α_v subunit in human embryonal kidney 293 cells. Because 293 cells express the vitronectin receptor α_vβ1 but have no endogenous β₃,39 transfection of β₃ into these cells enables them to mediate fibrin-clot retraction.24 The α_vLeu262Proβ₃complex expressed in stably transfected 293 cells had the same ligand specificity as α_IIbLeu262Proβ₃: 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 α_vLeu262Proβ₃ was likely due to quantitative differences in surface-expression levels or qualitative differences in the receptor-ligand interaction. Clot retraction mediated by α_vβ₃ and α_vLeu262Proβ₃ 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 Aα95-97 was not involved in α_IIbβ₃ binding to fibrinogen,41 and fibrinogen lacking the RGD motif at Aα572-574 still supported α_vβ₃-dependent clot retraction.42 We did not observe any inhibition by a fibrinogen γA 400-412 peptide, in keeping with reports that the γA region is also not required for α_IIbβ₃-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 α_IIbβ₃.12 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 β₃ was highly conserved among β 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 Cys273.30Limited tryptic proteolysis of α_IIbβ₃ and N-terminal sequencing of the resulting fragments found β₃residues 217 to 298 associated with α_IIbH residues 91-139 and α_IIbL residues 1 to 26,43 suggesting that this region of β₃ is involved in intersubunit contacts. Therefore, the Leu262Pro substitution may affect complex conformation and stability by disrupting a β₃ contact site for α_IIb. A mAb that recognizes β₃ residues 262 to 302, P₄₀, was also characterized.44 P₄₀was reported to bind platelet α_IIbβ₃only after treatment with EDTA, whereas it could bind endothelial cell α_vβ₃ without receptor dissociation.44 This implies, first, that in α_IIbβ₃, the β₃ 262-302 epitope is not surface exposed, consistent with the hypothesis that it forms part of an interface with α_IIb, and second, that the conformation of β₃ in α_IIbβ₃ differs from that of β₃ complexed to α_v. A comparison of the poor expression levels of α_IIbLeu262Proβ₃in both platelets and COS cells with the high expression of α_vLeu262Proβ₃ in 293 cells indeed indicates that the conformation of the 232-273 loop is more critical for association with α_IIb than with α_v.

Sequences immediately adjacent to the 232-273 loop of β₃have been implicated in ligand binding. Several research groups have observed that β₃ peptides corresponding to residues 211 to 231 can inhibit fibrinogen binding to α_IIbβ₃,17,45,46 but they disagreed about whether the peptide bound fibrinogen17,45or interacted with α_IIbβ₃itself.46 One study reconciled these viewpoints by reporting that the β₃ peptide 214-218 binds α_IIbβ₃, whereas a 217-231 peptide binds fibrinogen.47 In addition, structural modeling and mutagenesis of β₃ identified both Asp217 and glutamic acid 220 as potential cation-coordinating residues in the MIDAS-like domain of β₃.7,48 On the C-terminal side, a recombinant fragment comprising β₃ residues 274 to 368 was found to bind to the γ chain of fibrinogen and a mAb against residues 274 to 403 inhibited fibrinogen binding to platelets, suggesting that this region of β₃ 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 α_IIbβ₃-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 α_IIbβ₃ 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 α_IIbβ₃ 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 γ-chain C-terminal.12 13 The in vitro phenomenon of clot retraction and the ability of certain thrombasthenic α_IIbβ₃ mutants to retain fibrin binding provide a powerful approach for exploring the α_IIbβ₃-fibrin interaction. Additional selective mutagenesis studies of β₃ residues 232 to 273 based on the above findings may clarify the regulatory role of this intrachain loop in receptor activation.

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.