A Gln747→Pro Substitution in the _IIb Subunit Is Responsible for a Moderate _IIbβ₃Deficiency in Glanzmann Thrombasthenia

INTEGRIN α_IIbβ₃ (platelet GPIIb-IIIa), a calcium-dependent heterodimeric complex, is a prototype integrin that functions as a physiologic receptor for fibrinogen and von Willebrand factor and plays a crucial role in normal hemostasis and platelet aggregation.1-3 The importance of this integrin has been well documented by the clinical features of Glanzmann thrombasthenia (GT), a rare autosomal recessive bleeding disorder characterized by a quantitative or qualitative abnormality of α_IIbβ₃.4 

Analysis of cultured human leukemic and megakaryocytic cell lines has led to a better understanding of the key steps for α_IIbβ₃ biosynthesis.5-7 The α_IIb subunit is synthesized as a single-chain precursor, proα_IIb, that associates with the β₃subunit within the endoplasmic reticulum of cells. The proα_IIbβ₃ complex is then transported to the Golgi apparatus, where α_IIb undergoes sugar modification and endoproteolytic cleavage into heavy and light chains. After these processing events within the Golgi apparatus, the mature α_IIbβ₃ complex is rapidly transported to the cell surface. Classically, GT can be divided into three subgroups according to the amount of α_IIbβ₃: type I has a severe α_IIbβ₃ deficiency (<5% of normal), type II has a moderate α_IIbβ₃deficiency (10% to 20% of normal), and a variant has normal to near normal levels of a dysfunctional α_IIbβ₃(50% to 100% of normal).4 To date, more than 30 mutations in either the α_IIb or β₃ gene responsible for the thrombasthenic phenotype have been identified.8,9However, most of the reported mutations are responsible for severe α_IIbβ₃ deficiency (type I GT). Among these, the single amino acid substitutions have been especially informative in defining precise structural domains of integrins that play a role in the biosynthesis and/or function. For example, Gly242 → Asp (Gly273 → Asp)10 and Gly418 → Asp11 in α_IIb have been characterized in type I GT; these were highly conserved residues adjusted to the first calcium binding domain and flanking the fourth calcium binding domain of α_IIb, respectively. By contrast, the molecular basis for moderate α_IIbβ₃ deficiency (type II GT) remains obscure. Four mutations have been reported: Leu183 → Pro12 and Arg327 → His13,14 in α_IIb; Leu117 → Trp15 and Cys374 → Thr16 in β₃.

We have recently demonstrated that the amount of α_IIb is much lower than that of β₃ in a number of Japanese GT patients.17 Our data suggest that the molecular defect may exist more often in the α_IIb gene than in the β₃ gene in Japanese GT patients. In this study, we describe a new single amino acid substitution (Gln747 → Pro) in α_IIb responsible for moderate α_IIbβ₃ deficiency in 4 unrelated GT patients. Among them, patient MT (type II) was homozygous for the Gln747 → Pro substitution and patient MS (type I) was compound heterozygous for this substitution and a RNA splice mutation.

Patient MT, the product of nonconsanguineous parents, was a 40-year-old Japanese woman who had a life-long history of moderate mucocutaneous bleeding. Hematological examinations showed a prolonged bleeding time and absence of platelet aggregation in response to ADP, epinephrine, and collagen, but a normal response to ristocetin. Clot retraction was normal. She was patient no. 7 in our previous report and was classified as type II GT.17 Patient MS, the product of nonconsanguineous parents, was a 44-year-old Japanese woman who was also diagnosed as a typical case of GT. Clot retraction was slightly impaired (38%; normal values, 48% to 68%). Patients MT and MS were unrelated.

Rabbit polyclonal antisera specific for α_IIbβ₃ and murine monoclonal antibodies (MoAbs) AP2 (α_IIbβ₃-specific MoAb) were generously provided by Dr Thomas J. Kunicki (Scripps Research Institute, La Jolla, CA).18 AP3 (β₃-specific MoAb) was a generous gift from Dr Peter Newman (The Blood Center of Southeastern Wisconsin, Milwaukee, WI).19 PAC-1 (a ligand mimetic MoAb) binds specifically to activated α_IIbβ₃ and was kindly provided by Dr Sanford Shattil (Scripps Research Institute).20 PT25-2 (α_IIbβ₃-specific MoAb) activates α_IIbβ₃ and was a kind gift from Drs Makoto Handa and Yasuo Ikeda (Keio University, Tokyo, Japan).21TP80 (α_IIb-specific MoAb) and MOPC21 were purchased from Nichirei (Tokyo, Japan) and Sigma Chemical (St Louis, MO), respectively.

FK633(N-(N-{4-(4-Amidinophenoxy)butyl}-a-L-aspartyl-L-valine), a peptidomimetic antagonist specific for α_IIbβ₃, was generously provided by Dr Jiro Seki (Fujisawa Pharmaceutical Co, Osaka, Japan).22 

Immunoblot assay using rabbit polyclonal antisera specific for α_IIbβ₃ and flow cytometric analysis using various MoAbs were performed as previously described.23 24The amount of α_IIb and β₃ was semiquantified by densitometry using a CS 9000 dual-wavelength flying spot scanner (Shimadzu Corp, Kyoto, Japan).

Total cellular RNA of platelets was isolated from 30 mL of whole blood and α_IIb or β₃ mRNA was specifically amplified by reverse transcription-polymerase chain reaction (RT-PCR), as previously described.24 The primers for the amplification of α_IIb mRNA and conditions for RT-PCR were described elsewhere.24 The following primers were constructed based on the published sequence of β₃²⁵ and used for the first-round PCR of β₃ mRNA: IIIa1, 5′-CGGCCCCGGCCGCTCTGGGTGACTG-3′ (sense, nucleotide [nt] −15-10); IIIa2, 5′-CAACTCTTCAGGGAGGTCACG-3′ (antisense, nt 1147-1127); IIIa3, 5′-GAGCTCATCCCAGGGACCAC-3′ (sense, nt 1015-1034); and IIIa4, 5′-CACTGACTCAATCTCGTCACGGC-3′ (antisense, nt 1974-1952). IIIa7 and IIIa8 were described elsewhere.24 The following nested primers were used for the second-round PCR: IIIa1-Sal I, 5′-CTGTCGACGCGCTGGGGGCGCTG-3′ (sense, nt 8-30; mismatched sequences were underlined); IIIa2-Sph I, 5′-GGGCATGCACGCACTTCCAGCTC-3′ (antisense, nt 1137-1114); IIIa3-Sal I, 5′-GGGTCGACAGTTGGGGTTCTGTC-3′ (sense, nt 1027-1049); IIIa4-Sph I, 5′-GACGCATGCTCGTCACGGCAGTAACG-3′ (antisense, nt 1945-1970); IIIa7-Sal I, 5′-CTAGTCGACCAATGGGCTGCTGTG-3′ (sense, nt 1749-1772); and IIIa8-Sph I, 5′-GGCGCATGCTGATAATGATCTGAG-3′ (antisense, nt 2376-2353).

Nucleotide sequences of PCR products and subcloned cDNA fragments were determined by using Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).

Amplification of the region around exon 23 of the α_IIbgene was performed by using primers IIbE23, 5′-CAGGTCTAACTTCAGTGTGGC-3′ (sense, nt 13134-13154 in the α_IIb gene), and IIbE24, 5′-CAGGATGTAGAGCAGGTC-3′ (antisense, nt 13761-13744), using 250 ng of DNA as a template.26 The first-round PCR products were reamplified using primers IIbE23 and IIbE24Pvu II, 5′-CTCTCACCCTCGCAGCTCAGCT-3′ (antisense, nt 13355-13334; mismatched sequences were underlined). PCR products were then digested with restriction enzyme Pvu II. For the amplification of the region around exon 18 of the α_IIb gene, amplified DNA fragments using primers IIbE16, 5′-GAGGTCGACTTACGTCTTTTGC-3′ (sense, nt 9324-9344), and IIbI18, 5′-GGGTTACATTGTGACTTGGCAC-3′ (antisense, nt 10048-10027), were reamplified using nested primers IIbE17A, 5′-ATGCCGAGCTGCAGCTG-3′ (sense, nt 9501-9517), and IIbI18. PCR products were digested with Avr II. The resulting fragments were electrophoresed in a 6% polyacrylamide gel.

The α_IIb and β₃ cDNA constructs were cloned into a mammalian expression vector pcDNA3 (Invitrogen Corp, San Diego, CA) and generously provided by Dr Peter Newman. To construct the expression vectors containing the ²³³⁴A (wild-type [WT]) or ²³³⁴C (Pro747) form of α_IIb cDNA, PCR-based cartridge mutagenesis was performed. The 1,184-bp region (nt 1988-3171) of platelet α_IIb cDNA from patient MS, who was heterozygous for ²³³⁴A and ²³³⁴C, was amplified by RT-PCR using primers IIb5 and IIb8. Then, second-round amplification was performed using 1 μL of the first-round PCR products as a template with nested primers IIb5A, 5′-CCAGATAGGAATCGCGATG-3′ (sense, nt 2185-2203), and IIb8Xba I, 5′-CCTTCTAGAATAGTGTAGGCTGCACC-3′ (antisense, nt 3148-3123; mismatched sequence was underlined) and Vent Polymerase (New England Biolabs, Beverly, MA). The amplified fragments were digested withRsr II and Xba I, and the resulting 823-bp fragments (nt 2318-3140) were extracted using GeneClean II kit (Bio 101, La Jolla, CA). The 2,367-bp fragment extending from the beginning of the open reading frame to nt 2317 was obtained by digesting the full-length of α_IIb cDNA with HindIII andRsr II. These two fragments were double-inserted into the pcDNA3 digested with HindIII and Xba I. Single clones that encode A or C at nt 2334 were selected by PCR followed byPvu II digestion. The selected clones were characterized by sequence analysis to verify the absence of any other substitutions and the proper insertion of the PCR cartridge into the vector.

To generate an Ala747α_IIb construct, we performed the site-directed mutagenesis by PCR. We synthesized mismatched sense primer IIb747Ala, 5′-CCGGTCCGGGCAGAGGCCGCAGTG-3′ (sense, nt 2315-2338; mismatched sequences were underlined) and performed PCR using full-length α_IIb cDNA as a template and primers IIb747Ala and IIb8Xba I. PCR products were digested withRsr II and Xba I. The 823-bp fragments (nt 2318-3140) were shuttled into pcDNA3 as described above. The mutant clones were characterized by sequence analysis to verify the absence of any other substitutions and the proper insertion of the PCR cartridge into the vector.

The wild-type or mutant α_IIb construct was cotransfected into 293 cells with wild-type β₃ construct by the calcium phosphate method, as previously described.27 The cells were cultured in Dulbecco’s modified medium (DME) with 10% heat-inactivated fetal calf serum (FCS).

Surface proteins of the transfected cells were biotinylated, and immunoprecipitation using MoAbs was performed as previously described.27 

Metabolic labeling of transfected cell was performed 1 day after transfection, as previously described.27 The cells were incubated with 0.2 mCi/mL of [³⁵S]-methionine for 120 minutes. For pulse chase study, the cells were incubated with 0.4 mCi/mL of [³⁵S]-methionine for 30 minutes and the medium was then changed to DME/10% FCS with 50 μg/mL of nonradioactive methionine. Cells were equally divided into five dishes and chased after 0, 2, 4, 8, and 24 hours, respectively. Immunoprecipitation was performed as previously described.27 

We first analyzed platelet proteins from patients MT and MS in an immunoblot assay under nonreducing (not shown) and reducing conditions (Fig 1). Various amounts of platelet proteins obtained from three normal subjects were also examined to obtain a standard curve. In patient MT, the amounts of α_IIb and β₃ were 15% and 22% of control, respectively, whereas in patient MS, α_IIb and β₃ were 4% and 8%, respectively. Abnormal α_IIb or β₃, such as a premature form of α_IIb, was not detected under nonreducing and reducing conditions in either patient. From these data, MT was classified as type II GT and MS as type I GT.

To identify the molecular defect in patients MT and MS, platelet mRNA was isolated from these patients and normal controls. The whole coding regions of α_IIb and β₃ cDNA were amplified by RT-PCR. Examination of nucleotide sequences of the PCR fragments using an ABI 373A DNA sequencer (Applied Biosystems, Foster City, CA) showed a single A → C substitution at nt 2334 in α_IIb cDNA that leads to a Gln747 → Pro substitution in exon 23 of α_IIb (Fig 2). Patient MT appeared homozygous for the ²³³⁴A → C substitution. The homozygosity of the substitution was confirmed by nucleotide sequence analysis of PCR fragments from genomic DNA (data not shown). No other nucleotide substitution was detected in either α_IIb or β₃ cDNA from patient MT. Patient MS was heterozygous for the ²³³⁴A → C substitution.

Because patient MS was heterozygous with severe deficiency, another genetic defect in the α_IIb gene was sought. Electrophoretic analysis of the first round of RT-PCR fragments using primers IIb3 and IIb4 in patient MS showed that two different-sized cDNAs were amplified: an expected size (1,031 bp) and a smaller size (∼900 bp) (Fig 3). Each cDNA fragment was subcloned into pUC19, and nucleotide sequences were analyzed. Sequence analysis of the smaller-sized fragment showed that a 126-bp region corresponding to the whole nucleotide sequence of exon 18 was deleted (Fig 3). No abnormality existed in the nucleotide sequence of the expected-sized fragment. The flanking region of exon 18 of the α_IIb gene was then amplified from genomic DNA of patient MS as well as control by PCR using primers IIbE16 and IIbI18. Nucleotide sequence showed an AG → AA substitution at the consensus splice acceptor site (−1) of exon 18 (Fig 3). No other nucleotide substitution was detected in either α_IIb or β₃ cDNA in patient MS. Thus, MS appeared to be heterozygous for the ²³³⁴A → C substitution and the G → A substitution at the splice acceptor site of exon 18 in the α_IIb gene.

To confirm that patient MS was a compound heterozygote, exon 23 and exon 18 with their flanking regions were amplified by PCR, followed by digestion with Pvu II and Avr II, respectively. A restriction site for Pvu II would be created by the²³³⁴A → C substitution and a restriction site forAvr II would be abolished by the G → A substitution. ASRA clearly indicated that the A → C substitution in exon 23 was derived from the patient’s father and that the G → A substitution at the splice acceptor site of exon 18 was derived from the mother (Fig4). These data confirmed that patient MS was a compound heterozygote. ASRA further confirmed that patient MT was homozygous for the A → C substitution in exon 23 (data not shown). Using ASRA, we examined the presence of the ²³³⁴A → C substitution in 15 other unrelated Japanese GT patients (type I, 8 cases; type II, 7 cases) and 20 control subjects. This substitution was present in 2 type II GT patients who were homozygote and heterozygote, respectively (data not shown). None of control subjects had this substitution.

To examine whether the ²³³⁴A → C substitution leading to Gln747 → Pro substitution (Pro747) in α_IIb might be responsible for type II GT, we constructed an expression vector that contained the wild-type or mutant Pro747 form of α_IIb. Each vector was cotransfected with the wild-type β₃ cDNA into 293 cells.

RNA blot analysis showed that the efficiency of transfection between the wild-type and the mutant Pro747α_IIb was essentially the same (data not shown). Flow cytometric analysis using the α_IIb-specific MoAb, TP80; the β₃-specific MoAb, AP3; and the α_IIbβ₃ complex-specific MoAb, AP2, showed that the level of mutant Pro747α_IIbβ₃ expression was moderately reduced compared with wild-type α_IIbβ₃expression (Fig 5). Immunoprecipitation of surface-labeled transfected cells using AP2 MoAb also showed that the amount of Pro747α_IIbβ₃ complex was moderately reduced compared with wild-type and that the molecular weight of the mutant α_IIb was the same as the wild-type (Fig 6A). Interestingly, in the mutant Pro747α_IIbβ₃ transfected cells, a significant amount of a premature form of α_IIb(proα_IIb) was precipitated by AP2 MoAb. These data indicate that proα_IIb could be expressed and complexed with β₃ on the surface of the mutant Pro747α_IIbβ₃ transfected cells. Densitometric analysis showed approximately 20% of normal levels of α_IIb (proα_IIb + α_IIb) and approximately 29% of normal levels of β₃ expressed on the surface of the Pro747α_IIbβ₃transfectants (mean of 2 separate experiments). Employing immunoblot assay using polyclonal antisera specific for α_IIbβ₃, we also examined the amount of α_IIbβ₃ in transfected cells. Again, the mature forms of Pro747α_IIb and β₃ in mutant transfected cells were moderately reduced compared with wild-type transfected cells (≈30% of normal levels of α_IIb, ≈48% of normal levels of β₃, n = 2; Fig 6B). However, the amount of proα_IIb was not reduced in mutant transfected cells (≈100% of normal levels of proα_IIb, n = 2; Fig 6B). These data indicate that the ²³³⁴A → C substitution leads to moderate reduction in the amount of α_IIbβ₃ within the transfected cells as well as on the cell surface.

To elucidate the mechanism of impaired expression of the mutant α_IIbβ₃, we examined the association between the mutant Pro747proα_IIb and β₃. Transfected cells were labeled with [³⁵S]-methionine for 2 hours; immunoprecipitation using TP80 MoAb or AP3 MoAb was then performed. Densitometric analysis of the immunoprecipitate showed that the β₃/(proα_IIb + mature α_IIb) ratios were essentially the same between wild-type and the mutant transfected cells. They were 0.66 (wild-type) and 0.67 (mutant) using TP80 and 1.93 (wild-type) and 1.91 (mutant) using AP3 (n = 2; Fig 7A). These results demonstrated that the association of the mutant proα_IIbwith β₃ was the same as that of wild-type proα_IIb. The densitometric analysis also showed that wild-type and the mutant transfected 293 cells synthesized β₃ in excess compared with proα_IIb and that approximately 70% of labeled β₃ was still in the free form.

The fate of the recombinant proteins was further examined in pulse-chase experiments. First, we examined the stability of the mutant Pro747proα_IIb. The α_IIb transfected cells were pulsed with [³⁵S]-methionine for 30 minutes, chased with unlabeled methionine for various periods of time, and then immunoprecipitated using TP80 MoAb. As shown in Fig 7B, Pro747 mutation did not affect the stability of the proα_IIb subunit. Next, to examine the effect of this mutation on the kinetics of α_IIbβ₃ complex formation, wild-type or Pro747α_IIb was cotransfected with wild-type β₃. Pulse-chase experiments showed that the mutant proα_IIb was clearly detected at 24 hours postchase and was more stable than wild-type proα_IIb when assembled with β₃. The mutant Pro747proα_IIb was cleaved into heavy and light chains. However, this process was moderately impaired compared with wild-type proα_IIb (Fig7C and D).

To further examine the role of the Gln residue at amino acid 747 of α_IIb on α_IIbβ₃ expression, we introduced a Gln747 → Ala mutation (Ala747) by PCR-based site-directed mutagenesis. The mutant Ala747 form of α_IIbcDNA was cotransfected with wild-type β₃ cDNA into 293 cells. Flow cytometric analysis using AP2 MoAb showed that the level of surface expression of Ala747α_IIbβ₃ complex on the transfected cells was almost the same as wild-type α_IIbβ₃ complex (mean fluorescence intensity: 81.9 for wild-type and 92.3 for Ala747α_IIbβ₃, n = 2; Fig8). These data indicate that the Gln747 → Ala mutation does not impair α_IIbβ₃expression.

Because the mutant Pro747α_IIbβ₃ receptors were expressed at substantial levels on the surface of transfected cells, we then examined the binding of the ligand-mimetic MoAb PAC-1 in the presence of the activating MoAb PT25-2. Negative control for the PAC-1 binding was obtained using FK633, a peptidomimetic antagonist specific for α_IIbβ₃. As shown in Fig 8, PAC-1 could bind to both Pro747α_IIbβ₃ and Ala747α_IIbβ₃ in the presence of PT25-2. The PAC-1 binding to activated α_IIbβ₃ was dependent on the PT25-2 binding and the PAC-1/PT25-2 binding ratios were 1.28 and 0.97 for Pro747α_IIbβ₃ and Ala747α_IIbβ₃, respectively, which were normalized relative to the ratio for wild-type. These data suggest that ligand binding function of Pro747α_IIbβ₃ and Ala747α_IIbβ₃ is not disturbed.

In this report, we described a new point mutation (²³³⁴A → C) leading to Gln747 → Pro amino acid substitution in α_IIb that is responsible for moderate α_IIbβ₃ deficiency (type II phenotype) in 6 of the 34 possibly mutant chromosomes in 17 unrelated Japanese GT patients. In addition, a G → A mutation at the consensus sequence of the splice acceptor site of exon 18 of the α_IIb gene that is likely to be responsible for the exon 18 skipping in α_IIb cDNA was also found. The exon 18 skipping leads to an in-frame deletion of 42 amino acids in the extracellular domain of α_IIb. Together with the Pro747 mutation, the deletion of exon 18 contributed to the severe reduction in α_IIbβ₃ expression (in type I GT patient MS).

We demonstrated that the Pro747 substitution in α_IIb was not a naturally occurring polymorphism of α_IIb. ASRA showed that none of 20 control subjects possessed this substitution. Mammalian expression vectors encoding the mutant Pro747 form of α_IIb were constructed and cotransfected with wild-type β₃ cDNA into 293 cells. Both flow cytometric and immunoprecipitation analysis using anti-α_IIbβ₃ MoAbs demonstrated that the Pro747 substitution directly leads to moderate reduction in α_IIbβ₃ expression on the cell surface (20% to 30% of wild-type). The impairment of reactivity with a panel of MoAbs was not due to disruption of their epitopes, because immunoblot analysis using polyclonal anti-α_IIbβ₃antisera clearly showed reduction in the total amount of α_IIbβ₃ in transfected cells. Recently, it has been demonstrated that the Leu183 → Pro mutation in α_IIb leads to both quantitative and qualitative abnormalities in α_IIbβ₃.12However, the Gln747 → Pro mutation did not impair the ligand-binding function. These results demonstrate that the Pro747 mutation only leads to a quantitative abnormality.

Characterization of the two mutations (Asp242 and Asp418) flanking the calcium-binding domains of α_IIb responsible for type I GT indicates that these relatively well-conserved flanking sequences are not essential for assembly of the α_IIbβ₃heterodimer; rather, they are critical for the proper folding of α_IIbβ₃ to be transported to the Golgi apparatus, where α_IIb cleavage occurs.10,11However, the Pro747 mutation was far from the calcium-binding domains. By means of protein analysis of the proteolytic fragments of isolated α_IIbβ₃ heterodimers, Calvete et al28 have demonstrated that three regions of the α_IIb heavy chain were involved in interaction with β₃: amino acids 486-553, 696-734, and 780-814. Because amino acid 747 was close to the 696-734 region, we examined whether the Pro747 mutation might impair assembly of α_IIbβ₃ heterodimers. Immunoprecipitation analysis using metabolically labeled transfected cells clearly indicated that assembly of the mutant Pro747proα_IIb and β₃ normally occurred. In contrast to the Asp242 and Asp418 mutations, pulse chase studies demonstrated that some of the mutant Pro747proα_IIbβ₃ complexes underwent endoproteolytic cleavage of proα_IIb into heavy and light chains. These data suggest that some α_IIbβ₃heterodimers can be transported to the Golgi apparatus. Thus, the Pro747 mutation does not completely prevent intracellular transport of the heterodimers to the Golgi apparatus.

Recently, Kolodziej et al29 demonstrated that the endoproteolytic cleavage of proα_IIb occurs on the carboxyl side of dibasic Arg858-Arg859. They also demonstrated that the failure of cleavage does not prevent expression of α_IIbβ₃ on the cell surface. Immunoprecipitation using AP2 MoAb of biotin-labeled surface proteins showed that, in addition to mature α_IIb, a small amount of proα_IIb was expressed on the Pro747 mutant transfected cells. Pulse-chase studies demonstrated that the stability of the mutant Pro747proα_IIb was increased only when assembled with β₃. These data suggest that the mutant proα_IIb complexed with β₃ also impairs cleavage of α_IIb to some extent, probably due to the induction of a conformation in α_IIb that is less favorable to protease activity. Because the cleavage of proα_IIb into heavy and light chains is not critical for surface expression of α_IIbβ₃,29our data suggest that the Pro747 mutation impairs intracellular transport of α_IIbβ₃ to the Golgi apparatus as well. Although our transfection experiments demonstrated that the mutation led to the expression of a significant amount of proα_IIb on the transfected cells, proα_IIbwas not detected in platelets from patients MT and MS even in the immunoblot assay. This is probably due to differences between transfected cells and platelets that circulate for 10 days with no new mRNA being made. In contrast to our patients, Jung et al30 have reported a type I GT patient whose platelets contained no normal α_IIb, but did show a trace amount of a premature form of α_IIb in an immunoblot assay.30 One of the genetic defects of their patient was an RNA splicing mutation leading to skipping of exon 26 that contains the endoproteolytic cleavage site of α_IIb.31However, this abnormal α_IIb could not be expressed on the platelet surface. Because exon 26 skipping led to loss of 42 amino acids as well as the cleavage site, it is likely that the mutation altered the conformation of α_IIbβ₃heterodimer sufficient to prevent intracellular transport in their case.

We replaced Gln747 with Ala by mutagenesis to examine the role of Gln747 on α_IIbβ₃ expression. In contrast to the Pro747 mutation, Ala747 substitution did not impair α_IIbβ₃ expression. These data suggest that the presence of Pro747, rather than the absence of Gln747, is critical for moderately impaired α_IIbβ₃ expression. Pro is an uncharged amino acid known to disrupt secondary protein structures, and a number of naturally occurring mutations creating a Pro residue leading to impaired expression or function of proteins have been reported.12,32-34 In GT patients, it has been well documented that the Ser752 → Pro mutation in the cytoplasmic domain of β₃ leads to a variant GT phenotype.32Pro752 mutation makes α_IIbβ₃ incapable of being activated by intracellular signals and decreases its capacity to mediate cell spreading.35,36 However, replacement of Ser752 with Ala had no adverse effects on α_IIbβ₃-mediated cell spreading.37 Kahn et al38 have demonstrated that replacement of Gly242 residue with the nonpolar amino acids Ala or Val had no effect on α_IIbβ₃ expression, whereas replacement with the negatively charged Glu, positively charged Lys, or nonpolar Pro caused intracellular retention of α_IIbβ₃.

In summary, we have described a novel point mutation, Gln747 → Pro in α_IIb responsible for type II GT. The mutation moderately impaired the intracellular transport of α_IIbβ₃ heterodimers to the Golgi apparatus and endoproteolytic cleavage of proα_IIb into heavy and light chains. Our in vitro studies suggest that the impairment of α_IIbβ₃ is likely due to the presence of the Pro747 residue, rather than to the absence of Gln747. Molecular genetic examination of additional GT patients should provide further insight into the structural requirements for α_IIbβ₃expression, as well as differences between type I and type II GT phenotypes.

The authors thank Dr Thomas J. Kunicki for the rabbit polyclonal antisera specific for α_IIbβ₃ and MoAb AP2; Dr Peter Newman for MoAb AP3 and the α_IIb and β₃ cDNA cloned into a mammalian expression vector pcDNA3; Dr Sanford Shattil for MoAb PAC-1; and Drs Makoto Handa and Yasuo Ikeda for MoAb PT25-2.