Missense mutations in the β₃ subunit have a different impact on the expression and function between α_IIbβ₃ and α_vβ₃

Integrins are a family of cell surface molecules that mediate cellular attachment to the extracellular matrix and cell cohesion and are involved in such diverse biologic processes as thrombus formation, angiogenesis, inflammation, and embryogenesis.1 Integrins are αβ heterodimers, and β₃ is one of 8 known β subunits. α_IIbβ₃ and α_vβ₃belong to the β₃ integrin subfamily and share the same β subunit (β₃).2,3α_IIbβ₃, whose expression is restricted to the megakaryocyte/platelet lineage, is a prototypic integrin that functions as a physiologic receptor for fibrinogen and von Willebrand factor and plays a crucial role in normal hemostasis and platelet aggregation.4 On the other hand, α_vβ₃ is expressed in a number of tissues, such as platelets, endothelial cells, smooth muscle cells, and osteoclasts, and plays a key role in cell proliferation, cell migration, angiogenesis, and bone resorption.5-7 

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder characterized by a quantitative or qualitative abnormality of α_IIbβ₃ and caused by a defect in either theαIIb or β₃ gene.8-11The quantitative abnormality in GT can be divided into 2 groups: type I has a severe α_IIbβ₃ deficiency (< 5% of normal) with no or minimal clot retraction, and type II has a moderate α_IIbβ₃ deficiency (10%-20% of normal) with normal or only moderately diminished clot retraction.8 The numbers of α_IIbβ₃ and α_vβ₃expressed on the platelet surface are 40 000 to 80 000 molecules per platelet and about 100 molecules per platelet, respectively.12 Previous studies have shown that α_IIbβ₃ and α_vβ₃are synthesized by a similar mechanism.13 The αIIb αv and β₃ subunits are synthesized from separate messenger RNA transcripts, and the β₃ subunit becomes associated with either proαIIb or proαv, single-chain precursor forms of α subunits, in the endoplasmic reticulum. The proα_IIbβ₃ and proα_vβ₃ complex are then transported to the Golgi apparatus, where proα subunits undergo sugar modification and endoproteolytic cleavage into heavy and light chains. After these processing events within the Golgi apparatus, the mature α_IIbβ₃ and α_vβ₃complex is rapidly transported to the cell surface.13,14Consistent with these biosynthetic processes, GT patients with mutations in the β₃ gene that cause impaired synthesis of β₃ are deficient in both α_IIbβ₃ and α_vβ₃, while patients with mutations in theαIIb gene are deficient only in α_IIbβ₃ and have normal or even increased α_vβ₃ on their platelets.12Thus, the level of α_vβ₃ expression appears to be a useful marker to differentiate patients with a genetic defect located in the β₃ gene and those in theαIIb gene_.12,15 However, it remains obscure whether missense mutations in the β₃ subunit may induce the same defects in both β₃ integrins.

In this study, we examined the effects of several β₃missense mutations, including a His280Pro mutation, on the expression and function of these β₃ integrins.

Rabbit polyclonal antisera specific for α_IIbβ₃ and AP2 (α_IIbβ₃-specific monoclonal antibody [MoAb]) were generously provided by Dr Thomas J. Kunicki (The Scripps Research Institute, La Jolla, CA).16 AP3 (β₃-specific MoAb) was generous gift from Dr Peter Newman (The Blood Center of Southeastern Wisconsin, Milwaukee, WI).17 PAC-1 (a ligand mimetic MoAb) binds specifically to activated α_IIbβ₃ and was kindly provided by Dr Sanford Shattil (The Scripps Research Institute).18PT25-2 (α_IIbβ₃-specific MoAb) activates α_IIbβ₃ and was a kind gift from Drs Makoto Handa and Yasuo Ikeda (Keio University, Tokyo, Japan).19LM609 (α_vβ₃ complex–specific MoAb) and LM142 (αv-specific MoAb) were generously provided by Dr David Cheresh (The Scripps Research Institute).20 TP80 (αIIb-specific MoAb) and MOPC21 (mouse myeloma immunoglogulin [Ig] G₁) were purchased from Nichirei (Tokyo, Japan) and Sigma Chemical (St Louis, MO), respectively. RGDW (Arg-Gly-Asp-Trp) peptide and FK633 (peptidomimetic antagonist specific for α_IIbβ₃) were generously provided by Dr Jiro Seki (Fujisawa Pharmaceutical, Osaka, Japan).21Cyclo(RGDfV) (cyclo(-Arg-D-Gly-D-Asp-D-Phe-L-Val-D-)) peptide specific for α_vβ₃ was a generous gift from Merck (Darmstadt, Germany).22 

Patient Osaka-5, a product of nonconsanguineous parents, was a 33-year-old Japanese woman who was diagnosed as a typical GT. Clot retraction by MacFarlane's method was normal (40%; normal values 40%-60%). An immunoblot assay using rabbit polyclonal antisera specific for α_IIbβ₃23 revealed that the amounts of α_IIb and β₃ in platelets from patient Osaka-5 were 6% and 8% of control platelets, respectively (data not shown). Although the amounts of α_IIbβ₃ in Osaka-5 did not fulfill the criteria for type II GT (10%-20% of normal), normal clot retraction of Osaka-5 platelets strongly suggested that she was classified as type II rather than type I GT.

Flow cytometric analysis using various MoAbs and immunoblot assay using rabbit polyclonal antisera specific for α_IIbβ₃ were performed as previously described.23,24 To examine the expression of α_vβ₃ on platelets, Alexa-conjugated goat F(ab′)₂ antimouse IgG (Molecular Probes, Eugene, OR) was used instead of fluorescein isothiocyanate (FITC)–conjugated goat antimouse IgG because of its higher sensitivity.

Quantitative enzyme-linked immunosorbent assay (ELISA) was performed to examine the amounts of α_vβ₃ in platelet lysates from control subjects or patient Osaka-5. In brief, 1 × 10⁶/μL washed platelets were solubilized in 0.05 M Tris-buffered saline, pH 7.4, containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 100 μg/mL leupeptin (Sigma). After centrifugation at 10 000g for 10 minutes, 100 μL lysate was applied to the wells of a microtiter tray, each containing 0.25 μg fixed LM609. After incubation for 60 minutes the tray was washed 6 times, and biotinylated LM142 was added to each well for 60 minutes. After washing 6 times, the bound LM142 was detected using an avidin–biotin–alkaline phosphatase complex (Vector, Burlingame, CA) and ELISA amplification system (Life Technologies, Gaithersburg, MD). Standard curve was obtained using purified α_vβ₃ purchased from Chemicon International (Temecula, CA).

Total cellular RNA of platelets was isolated from 30 mL of whole blood, and αIIb or β₃ messenger RNA was specifically amplified by reverse transcription–polymerase chain reaction (RT-PCR), as previously described.25 The primers for the amplification of αIIb or β₃ messenger RNA and conditions for RT-PCR were described elsewhere.25,26Nucleotide sequences of PCR products were determined by using Taq DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA).

Amplification of the region around exon 5 of theβ₃ gene was performed by using primers IIIaE5, 5′-CTCTACCAGTGACATGGCTG-3′ (sense, nucleotide [nt] 17 365-17 384 in the β₃ gene), and IIIaE6, 5′-GCCAGAGATCTCACCATGG-3′ (antisense, nt 17 607-17 589) using 250 ng DNA as a template.27 The first-round PCR products were reamplified using primers IIIaE5 and IIIaE6BspHI, 5′-CATGGTAGTGGAGGCAGAGTCA-3′ (antisense, nt 17 593-17 572, mismatched sequence underlined). PCR products were then digested with restriction enzyme BspHI. The resulting fragments were electrophoresed in a 6% polyacrylamide gel.

The wild-type αIIb and β₃ complementary DNAs (cDNAs) cloned into a mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA) were generously provided by Dr Peter Newman (Milwaukee, WI). The full-length αv cDNA was generously provided by Dr David Cheresh (La Jolla, CA) and shuttled into pcDNA3. To construct the expression vectors containing the 887C (Pro280) form of β₃ cDNA, PCR-based cartridge mutagenesis was performed. The 1654–base pair (bp) region (nt 350-2003) of platelet β₃ cDNA from patient Osaka-5, who was homozygous for 887C, was amplified by RT-PCR using primers IIIa1A, 5′-CCATCCAAGTGCGGCAGGTGG-3′ (sense, nt 350-370), and IIIa8AflII, 5′-GCATCCTTGCCAGTGTCCTTAAG-3′ (antisense, nt 2003-1981). The amplified fragments were digested with KpnI and AflII, and the resulting 1526-bp fragments (nt 456-1981) were extracted using GeneClean II kit (Bio 101, La Jolla, CA). The 530-bp fragments extending from the beginning of the open reading frame to nucleotide 455 were obtained by digesting the full-length of β₃ cDNA with BamHI and KpnI. These 2 fragments were double-inserted into the pcDNA3 digested withBamHI and AflII. The fragments inserted were characterized by sequence analysis to verify the absence of any other substitutions and the proper insertion of the PCR cartridge into the vector.

For the introduction of other missense mutations in β₃leading to Leu117→Trp, Ser162→Leu, Arg216→Gln, or Cys374→Tyr, we carried out the overlapping extension PCR, as previously described.26 For example, to generate the Leu117→Trp β₃ (Trp117β₃) mutant, we synthesized mismatched sense primer IIIa117Trp-s, 5′-GGACATCTACATCTGGATGG-3′ (nt 384-403, mismatched sequence underlined), and antisense primer IIIa117Trp-as, 5′-GACAGGTCCATCCAGTAGTAG-3′ (nt 410-390, mismatched sequence underlined). PCR was performed by using β₃ cDNA as a template and primers pcDNA3-s, 5′-GGCTAACTAGAGAACCCACTG-3′ and IIIa117Trp-as, or primers IIIa117Trp-s and IIIa1α 5′-GCGGGTCACCTGGTCAG-3′ (antisense, nt 654-648). The 2 individually amplified PCR products were mixed and used as a template of PCR using primers pcDNA3-s and IIIa1α. The amplified PCR products were digested with KpnI, and then the fragments were introduced into pcDNA3 as described above. The fragments inserted were characterized by sequence analysis.

Ten micrograms of wild-type or mutant β₃ construct was cotransfected into human embryonic kidney 293 cells (10⁶cells) with 10 μg wild-type αIIb or αv construct by the calcium phosphate method as previously described.28 The 293 cells transiently expressing α_IIbβ₃ or α_vβ₃ were obtained and analyzed 2 days after transfection. In selected experiments, 100 ng green fluorescent protein (GFP) expression vector pEGFP-C1 (Clontech, Palo Alto, CA) was cotransfected with β₃ and either αIIb or αv construct into 293 cells to monitor transfection efficiency. The cells were cultured in Dulbecco modified medium with 10% fetal calf serum.

Surface proteins of the transfected cells were biotinylated 2 days after transfection, and immunoprecipitation using MoAbs was performed as previously described.28 

Metabolic labeling of transfected cells was performed one day after transfection as previously described.25 The cells were incubated with 0.4 mCi/mL (14.8 MBq) [³⁵S]methionine for 30 minutes, and then the medium was changed to Dulbecco modified medium/10% fetal calf serum with 50 μg/mL nonradioactive methionine. Cells were equally divided into 3 dishes and chased after 0.5, 2, and 22 hours, respectively, and immunoprecipitation was performed.25 

Soluble fibrinogen binding assay was performed as previously described.29 For fibrinogen binding to α_vβ₃, 50 μL aliquots of α_vβ₃-transfected cells (1.5 × 10⁵) in Ca⁺⁺-free Tyrode-HEPES buffer containing 1 mM MgCl₂ were incubated with MoAb LM142 specific for αv (5 μg/mL) for 30 minutes on ice. After washing, 1 mM MnCl₂ was added into the cell suspension to induce a high-affinity state of α_vβ₃. Cells were then incubated with FITC-fibrinogen (150 μg/mL) in the presence or absence of 1 mM RGDW or 50 μM cyclo(RGDfV) (an α_vβ₃ antagonist) and phycoerythrin-conjugated antimouse IgG (1:5 dilution, Serotec, Oxford, United Kingdom) for 25 minutes at 22°C and then incubated with propidium iodine (Sigma) for 5 minutes at 22°C. After washing, fibrinogen binding (FL1) was analyzed on the gated subset of single, α_vβ₃-expressing (FL2) live cells (propidium iodine–negative, FL3). Specific fibrinogen binding was defined as that inhibited by 50 μM cyclo(RGDfV). For fibrinogen binding to α_IIbβ₃, α_IIbβ₃-transfected cells were examined in the presence or absence of 10 μM FK633 (an α_IIbβ₃ antagonist) with 1 mM CaCl₂ and 10 μg/mL PT25-2 (an α_IIbβ₃-activating antibody). The following procedures were the same as described above.

We examined the surface expression of α_IIbβ₃ and α_vβ₃on platelets from patient Osaka-5 using flow cytometry. While the GPIb-specific MoAb AP1 bound equivalently to Osaka-5 and control platelets, the α_IIbβ₃ complex–specific MoAb AP2, the β₃-specific MoAb AP3, and the αIIb-specific MoAb TP80 showed a marked reduction in the expression of α_IIbβ₃ on Osaka-5 platelets (Figure1A). The amount of α_IIbβ₃ expressed on Osaka-5 platelet surface was about 6% of control platelets (n = 11). On the other hand, Alexa-conjugated goat F(ab′)₂ antimouse IgG clearly showed that α_vβ₃ complex–specific MoAb LM609 reacted with Osaka-5 platelets as well as control platelets. The mean fluorescence intensity (MFI) for LM609 bound to control platelets was 1.56 ± 0.28 arbitrary units (mean ± SD, n = 11) and that to Osaka-5 platelets was 0.64 (mean of duplicates). Thus, the amount of α_vβ₃ expressed on Osaka-5 platelet surface appeared to be 41% of control platelets (Figure 1B). To further examine the expression of α_vβ₃ in Osaka-5 platelets, we measured the amounts of α_vβ₃in platelet lysates using sensitive ELISA. The amounts of α_vβ₃ in control platelets and in Osaka-5 platelets were 8.4 ± 2.1 ng/10⁸ platelets (n = 11) and 4.3 ng/10⁸ platelets, respectively (Figure 1C). These data demonstrated that α_vβ₃ expression in Osaka-5 platelets was about half as much as that in control platelets.

To identify the molecular defect in patient Osaka-5, the whole coding regions of αIIb and β₃ cDNAs were amplified by RT-PCR, as previously described.21 Examination of nucleotide sequences of the PCR fragments revealed that the β₃ cDNAs had a single A>C substitution at nucleotide 887 that leads to a His280→Pro substitution of β₃ (Figure2A). Patient Osaka-5 appeared homozygous for the 887A>C substitution, and no other nucleotide substitutions were detected in the coding regions of either αIIb or β₃ cDNAs from patient Osaka-5. To confirm that patient Osaka-5 was homozygous for the 887A>C substitution, exon 5 with their flanking regions of the β₃ gene were amplified by PCR, followed by digestion with BspHI. A restriction site for BspHI would be abolished by the 887A>C substitution. Allele-specific restriction enzyme analysis showed that Osaka-5 was homozygous for the A→C substitution in exon 5 (Figure 2B). The homozygosity of the substitution was also confirmed by nucleotide sequence analysis of the PCR fragments from genomic DNA (data not shown). Using allele-specific restriction enzyme analysis, we examined the presence of the 887A>C substitution in 18 other unrelated Japanese GT patients (type I, 8 cases; type II, 10 cases) and 20 control subjects. This substitution was also present in 2 other type II GT patients (1 homozygous, 1 heterozygous) (data not shown). No control subjects had this substitution.

We constructed an expression vector that contained the wild-type or the mutant Pro280 form of β₃ and cotransfected each β₃ construct with the wild-type αIIb construct into 293 cells. When 100 ng GFP expression vector was cotransfected, the levels of GFP expression were essentially the same between the wild-type α_IIbβ₃ and the mutant αIIbPro280β₃-transfected cells (Figure3A). Flow cytometric analysis using AP2 MoAb, AP3 MoAb, and TP80 MoAb showed that the level of the mutant αIIbPro280β₃ expression was markedly reduced compared with the wild-type α_IIbβ₃ expression (about 25% of wild-type, Figure 3A). Immunoprecipitation of the surface-labeled transfected cells using AP3 MoAb also showed that the amount of αIIbPro280β₃ complex was reduced and that the molecular weight of the mutant β₃ was the same as the wild-type (Figure 3B). Immunoblot assay using polyclonal antisera specific for α_IIbβ₃ revealed that the mature form of αIIb was more markedly reduced than β₃in the mutant transfected cells, as observed in Osaka-5 platelets (Figure 3C). To examine the effect of the His280→Pro substitution in β₃ on α_vβ₃ expression, we first transfected wild-type β₃ or the mutant Pro280β₃ construct into 293 cells. In these conditions, wild-type β₃ or the mutant Pro280β₃ could be associated with an endogenous human αv of 293 cells. The 293 cells transfected with empty vectors did not show any expression of α_vβ₃ (data not shown). Flow cytometric analysis using LM609 MoAb and LM142 MoAb showed that the level of surface expression of αvPro280β₃ complex on the transfected cells was almost the same as wild-type α_vβ₃ complex (Figure 3D). To rule out the possibility that the normal expression of α_vβ₃ was due to the presence of an excess amount of β₃, we transfected wild-type β₃or the mutant Pro280β₃ construct with wild-type αv construct into 293 cells. The cotransfection of αv and β₃ cDNAs into 293 cells markedly increased the level of α_vβ₃ expression. However, the surface expression of αvPro280β₃ complex was almost the same as wild-type α_vβ₃ complex (Figure 3D). In addition, reduction in the amount of the transfected β₃construct did not make any difference between the expression level of the wild-type and the mutant α_vβ₃ (data not shown). These data indicate that the 887A>C substitution leads to the marked reduction in the amount of α_IIbβ₃without disturbing α_vβ₃ expression at least in 293 cells.

To assess the ligand binding function of the mutant αIIbPro280β₃, we examined the binding of the ligand-mimetic MoAb PAC-1 in the presence of the activating MoAb PT25-2.25 PAC-1 could bind to the mutant αIIbPro280β₃ as well as the wild-type α_IIbβ₃ in the presence of PT25-2 (Figure3A). The PAC-1 binding to α_IIbβ₃ was dependent on the PT25-2 binding, and the PAC-1/PT25-2 binding ratio for αIIbPro280β₃ was essentially the same as that for wild type (PAC-1/PT25-2 ratio: wild type, 0.31 ± 0.12; mutant, 0.36 ± 0.14; mean ± SD; n = 3).

The different effects of the His280Proβ₃ mutation between α_IIbβ₃ and α_vβ₃ expression made us further examine the effects of other missense mutations found in GT patients on α_vβ₃ expression. Previously reported 4 single amino acid mutations in β₃ in GT were examined: Leu117Trp, Ser162Leu, Arg216Gln, and Cys374Tyr.30-33 In addition, we introduced a newly created Arg216Gln/Leu292Ser mutation into β₃. Each mutant β₃ cDNA vector was cotransfected with the wild-type αIIb cDNA or wild-type αv cDNA vector into 293 cells. Again, cotransfection of the GFP expression vector showed that the transfection efficiency was essentially the same between the wild-type and the mutant transfected cells (data not shown). As shown in Figure 4, we confirmed that all β₃ mutations examined markedly impaired surface expression of α_IIbβ₃. Immunoprecipitation of the surface-labeled α_IIbβ₃-transfected cells using AP3 further showed that the amounts of the mutant α_IIbβ₃ were markedly reduced compared with wild-type α_IIbβ₃ (Figure 4B). As shown in Figure 5, each of the Leu117Trp and Cys374Tyr mutations resulted in the marked reduction in α_vβ₃ expression as well. In sharp contrast, none of Ser162Leu, Arg216Gln, or Arg216Gln/Leu292Ser mutations impaired α_vβ₃ expression. Of particular interest was the effect of the Arg216Gln/Leu292Serβ₃ mutation. Although the Arg216Gln/Leu292Ser mutation completely abolished α_IIbβ₃ expression, this mutation did not impair α_vβ₃ expression at all. Because 293 cells possess endogenous αvβ1 and αvβ5,34,35 LM142 (anti-αv) may detect these αv integrins. However, our previous study showed that the expression of these endogenous αv integrins appeared to be low compared with exogenous α_vβ₃.34 Indeed, the Leu117Trp β₃ mutation severely impaired the expression of αv subunits as well as α_vβ₃, suggesting that the bulk of expressed αv integrins is α_vβ₃ in these conditions (Figure5).

To elucidate the mechanism of impaired expression of the mutant α_IIbβ₃, we performed pulse chase experiments especially for His280Pro and Arg216Gln/Leu292Ser mutations. Because β₃ was synthesized in excess as compared with αIIb in wild-type transfected cells in our experimental conditions,25 we used TP80 (anti-αIIb) and LM142 (anti-αv) for the precipitation of α_IIbβ₃and α_vβ₃ complex, respectively. As shown in Figure 6A, the association between proαIIb and Pro280β₃ or Gln216/Ser292β₃was the same as that of wild-type β₃ at 30 minutes after chase. At 2 hours after chase, some of the wild-type proα_IIbβ₃ complex was transported to the Golgi apparatus, where cleavage of proαIIb into heavy and light chains occurs. However, this process was impaired in His280Pro and Arg216Gln/Leu292Ser mutants. Even at 22 hours after chase, mature α_IIbβ₃ was not detectable in Arg216Gln/Leu292Ser mutant, while a small amount of mature α_IIbβ₃ was observed in the His280Pro mutant. In sharp contrast to α_IIbβ₃mutants, the kinetics of αvHis280Proβ₃ and αvArg216Gln/Leu292Serβ₃ biosynthesis were the same as that of wild type, and the normal amount of mature α_vβ₃ was synthesized at 22 hours after chase (Figure 6B).

We then assessed the ligand binding function of the mutant β₃ integrins. We measured the binding of FITC-fibrinogen to mutant α_IIbβ₃ and α_vβ₃. The α_IIbβ₃-transfected cells were treated with the α_IIbβ₃-activating antibody, PT25-2, while α_vβ₃-transfected cells were treated with 1 mM MnCl₂, which induces a high-affinity state of α_vβ₃. Although 1 mM MnCl₂ has also been shown to result in fibrinogen binding to α5β1 in endothelial cells,36 dot plots in Figure7B show that fibrinogen binding to the transfected 293 cells depend on the expression levels of α_vβ₃. In addition, the blockade of the fibrinogen binding by α_IIbβ₃-specific antagonist FK633 at 10 μM and α_vβ₃-specific antagonist cyclo(RGDfV) at 50 μM indicated that the binding was specifically mediated by α_IIbβ₃ and α_vβ₃, respectively, in our experimental conditions (Figure 7). Because the expression levels of α_IIbβ₃ and α_vβ₃were different in each mutation (Figures 4 and 5), we monitored α_IIbβ₃ and α_vβ₃expression by PT25-2 and LM142, respectively, and only analyzed the cells expressing the same levels of α_IIbβ₃and α_vβ₃ (Figure 7). Two variant type GT mutations in β₃, Asp119Tyr and Arg214Trp, were examined in parallel as negative controls.37,38 As expected, Asp119Tyr and Arg214Trp mutations abolished the ligand binding function of both β₃ integrins. Neither His280Pro nor Cys374Tyr mutation impaired the ligand binding to both α_IIbβ₃ and α_vβ₃. Ser162Leu mutation markedly impaired the ligand binding to α_IIbβ₃ but not to α_vβ₃ at all (Figure 7B). Similarly, Arg216Gln more severely impaired the ligand binding to α_IIbβ₃ than to α_vβ₃. Thus, Ser162Leu and Arg216Gln mutations showed a different effect on ligand binding between 2 β₃ integrins.

Among genetic defects responsible for GT phenotype, single amino acid substitutions in each subunit have been especially informative in defining precise structural domains of α_IIbβ₃ that play a role in the biosynthesis and/or function.9-11 However, it remains elusive whether missense mutations in β₃ responsible for GT may induce the same defects in the other β₃ integrin, α_vβ₃. In this study we investigated the effects of 6 missense mutations in β₃, including His280Pro mutation, on the expression and function of α_IIbβ₃ and α_vβ₃in 293 cells. Leu117Trp and Cys374Tyrβ₃ mutations impaired both α_IIbβ₃ and α_vβ₃ expression, while His280Pro, Ser162Leu, Arg216Gln, and Arg216Gln/Leu292Serβ₃ mutations impaired α_IIbβ₃ expression but not α_vβ₃ expression. With regard to ligand binding, Ser162Leu and Arg216Gln mutations markedly impaired the ligand binding to α_IIbβ₃ but not to α_vβ₃. Our present data demonstrate that some β₃ missense mutations have a different impact on the expression and function of α_IIbβ₃ and α_vβ₃.

The αIIb and αv are homologous and 36% identical in primary amino acid sequence.39 The αIIb subunit has been found only in combination with β₃, while αv is promiscuous and can associate with at least 5 β subunits (β1, β₃, β5, β6, and β8).2 As shown in Figure8, our data show that missense mutations at well-conserved Leu117 and Cys374 residues among 8 β subunits impaired the expression of both β₃integrins.40 In contrast, amino acid residues at positions 162, 216, 280, and 292 of β subunits are rather diverse, and mutations at these residues impaired only α_IIbβ₃ expression. Except for the Cys374Tyr mutation, His280Pro, Ser162Leu, and Arg216Gln mutations responsible for type II GT phenotype did not impair α_vβ₃expression in 293 cells, while the Leu117Trp mutation responsible for type I GT phenotype disturbed α_vβ₃expression. From these data one could argue that different effects of β₃ mutations on the biosynthesis of α_vβ₃ may reflect only the severity of α_IIbβ₃ deficiency. However, a newly created double mutation, Arg216Gln/Leu292Ser, clearly denied this possibility, because the mutation led to a severe α_IIbβ₃deficiency but normal α_vβ₃ expression. These findings strongly suggest that the expression of α_IIbβ₃ is more strictly regulated than α_vβ₃.

We found a point mutation (887A>C) leading to His280→Pro amino acid substitution in β₃ in 3 unrelated GT patients from 19 Japanese GT patients: 2 patients appeared homozygous, and 1 patient was heterozygous. Thus, this mutation was found in 5 of the 38 possibly mutant chromosomes. In addition to our patients, 3 other Japanese GT patients with this mutation have been reported.41 Although we could not rule out the possibility that patient Osaka-5 is hemizygous for the mutation, the prevalence of the His280Pro mutation in Japanese GT patients suggests that patient Osaka-5 is likely homozygous rather than hemizygous. Cotransfection of wild-type αIIb and Pro280β₃ constructs into 293 cells resulted in an impaired surface expression of α_IIbβ₃(about 25% of control). These data demonstrated that the His280Pro mutation is responsible for GT phenotype. It has been demonstrated that the hypothetical human β₃ metal ion–dependent adhesion site domain is critical for heterodimer assembly with human αIIb and ligand binding function.42,43 Moreover, the hexapeptide sequence 275Val-Gly-Ser-Asp-Asn-His280 within the β₃metal ion–dependent adhesion site domain appears necessary for species-restricted heterodimer formation.43 Our present data demonstrated that the His280Pro mutation at the sixth residue of the unique hexapeptide did not impair either assembly of proαIIb and β₃ or ligand binding function. In pulse chase studies, very little proαIIb was processed into mature αIIb, suggesting that at least a portion of this mutant proα_IIbβ₃ was retained and degraded within endoplasmic reticulum.

Because platelets express only a limited number of α_vβ₃ (about 100 per platelet), we carefully examined the expression levels of α_vβ₃ in Osaka-5 platelets. In sharp contrast to the markedly impaired α_IIbβ₃ expression (about 6% of normal), sensitive ELISA as well as flow cytometric analysis showed that Osaka-5 platelets possessed about 50% of the normal α_vβ₃ content, that is, an apparently higher amount of α_vβ₃ than was previously reported in GT platelets due to β₃ mutations (< 20% of normal).12 Our transient transfection studies may induce higher expression of the mutant β₃ integrins in 293 cells than in the patient's platelets, probably due to pcDNA3-derived overexpression of these proteins in heterologous cells (α_IIbβ₃, about 6% in Osaka-5 platelets vs about 25% in 293 cells; α_vβ₃, about 50% in Osaka-5 platelets vs about 100% in 293 cells). Nevertheless, our data clearly showed the different impact of the His280Proβ₃ mutation on the expression of the 2 β₃ integrins in Osaka-5 platelets as well as in 293 cells. Contrary to our findings, employing Chinese hamster ovary cells Ambo et al41 demonstrated that this mutation impaired the expression of β₃ when complexed with endogenous hamster αv. The difference in the expression of the mutant α_vβ₃ between human 293 and Chinese hamster ovary cells is likely due to a difference between species.

There are some distinctive features between α_IIbβ₃ and α_vβ₃. Treatment of α_IIbβ₃ with ethylenediaminetetraacetic acid at 37°C dissociates the complex into its individual subunits, while α_vβ₃ remains a heterodimer.44This difference may reflect tighter cation binding to α_vβ₃ or additional cation-independent interactions between αv and β₃. Divalent cations are also required to support ligand binding functions of the β₃ integrins. However, particular divalent cations affect ligand binding to the 2 receptors differently. Namely, fibrinogen binds to α_vβ₃ in the presence of Mn²⁺but not in Ca⁺⁺, while it binds to α_IIbβ₃ in either cation.45 In addition, we recently clarified the difference in the ligand binding sites between αIIb and αv.34 In this study, we newly demonstrate that Ser162Leu and Arg216Gln mutations show a different effect on ligand binding between 2 β₃ integrins. Consistent with the reports by Newman's group,32,33 we showed that Ser162Leu and Arg216Gln mutations impaired the stability of the complex between αIIb and β₃, as evidenced by the fact that the binding of complex-specific MoAb AP2 was markedly impaired compared with that of the αIIb-specific MoAb TP80. In contrast, neither mutation affected the stability of the complex between αv and β₃, as evidenced by the normal binding of the α_vβ₃ complex–specific MoAb LM609. These findings further indicate structural differences between α_IIbβ₃ and α_vβ₃. Employing αv/αIIb chimeras, it has been reported that ligand recognition specificity of α_IIbβ₃ is regulated by the amino-terminal one third of the α subunit that contains the amino-terminal 140 residues and first 2 divalent cation binding repeats of αIIb.46 Because missense mutations in β₃affect the expression and function of β₃ integrins differently, the key structure should lie in the α subunits. Further investigation of the structures in the α subunits that regulate the biosynthesis of the β₃ integrins is underway.

Leukocyte adhesion deficiency is a genetic disease characterized by abnormality of β₂ integrins.47-49 In leukocyte adhesion deficiency, missense mutations have been shown to impair expression of all β₂ integrins, α_Lβ₂ (CD11a/CD18), α_Mβ₂ (CD11b/CD18), and α_Xβ₂ (CD11c/CD18).50,51 There is so far no example of a selective deficiency in only 1 or 2 of the β₂ integrins.47 Because α_L, α_M, and α_X have been found only in combination with β₂, biosynthesis of α_Lβ₂, α_Mβ₂, and α_Xβ₂ may be regulated in a common mechanism. It should also be interesting to carefully examine whether some missense mutations in β₂ may affect the expression of these β₂ integrins differently.

In conclusion, we demonstrate that α_IIbβ₃and α_vβ₃ expression and function are differently regulated by certain β₃ missense mutations. We also suggest that Ser162 and Arg216 residues regulate the stability of α_IIbβ₃ and α_vβ₃ differently. These findings would provide insights into the structural requirement for α_IIbβ₃ and α_vβ₃function as well as their expression.

We thank Dr Thomas J. Kunicki for the rabbit polyclonal antisera specific for α_IIbβ₃ and for MoAb AP2; Dr Peter Newman for MoAb AP3 and the αIIb and β₃ cDNA cloned into a mammalian expression vector pcDNA3; Dr David Cheresh for MoAbs LM609 and LM142 and the αv cDNA cloned into a mammalian expression vector pcDNA1NEO; Dr Sanford Shattil for MoAb PAC-1; Drs Makoto Handa and Yasuo Ikeda for MoAb PT25-2; Dr Jiro Seki for FK633; and Dr P. Raddatz for cyclo(RGDfV).