αIIbβ3 and αvβ3 belong to the β3integrin subfamily. Although the β3 subunit is a key regulator for the biosynthesis of β3 integrins, it remains obscure whether missense mutations in β3 may induce the same defects in both αIIbβ3 and αvβ3. In this study, it is revealed that thrombasthenic platelets with a His280Pro mutation in β3, which is prevalent in Japanese patients with Glanzmann thrombasthenia, did contain significant amounts of αvβ3 (about 50% of control) using sensitive enzyme-linked immunosorbent assay. Expression studies showed that the His280Proβ3 mutation impaired αIIbβ3 expression but not αvβ3 expression in 293 cells. To extend these findings, the effects of several β3 missense mutations leading to an impaired αIIbβ3expression on αvβ3 function as well as expression was examined: Leu117Trp, Ser162Leu, Arg216Gln, Cys374Tyr, and a newly created Arg216Gln/Leu292Ser mutation. Leu117Trp and Cys374Tyr β3 mutations did impair αvβ3 expression, while Ser162Leu, Arg216Gln, and Arg216Gln/Leu292Ser mutations did not. With regard to ligand binding function, Ser162Leu mutation induced especially distinct effects between 2 β3 integrins: it markedly impaired ligand binding to αIIbβ3 but not to αvβ3 at all. These data clearly demonstrate that the biosynthesis and the ligand binding function of αIIbβ3 and those of αvβ3 are regulated in part by different mechanisms. Present data would be a clue to elucidate the regulatory mechanism of expression and function of β3 integrins.

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. αIIbβ3 and αvβ3belong to the β3 integrin subfamily and share the same β subunit (β3).2,3αIIbβ3, 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, αvβ3 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 αIIbβ3 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 αIIbβ3 deficiency (< 5% of normal) with no or minimal clot retraction, and type II has a moderate αIIbβ3 deficiency (10%-20% of normal) with normal or only moderately diminished clot retraction.8 The numbers of αIIbβ3 and αvβ3expressed 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 αIIbβ3 and αvβ3are 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 proαIIbβ3 and proαvβ3 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 αIIbβ3 and αvβ3complex 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 αIIbβ3 and αvβ3, while patients with mutations in theαIIb gene are deficient only in αIIbβ3 and have normal or even increased αvβ3 on their platelets.12Thus, the level of αvβ3 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.

Antibodies and antagonists

Rabbit polyclonal antisera specific for αIIbβ3 and AP2 (αIIbβ3-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 αIIbβ3 and was kindly provided by Dr Sanford Shattil (The Scripps Research Institute).18PT25-2 (αIIbβ3-specific MoAb) activates αIIbβ3 and was a kind gift from Drs Makoto Handa and Yasuo Ikeda (Keio University, Tokyo, Japan).19LM609 (αvβ3 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 αIIbβ3) 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 αvβ3 was a generous gift from Merck (Darmstadt, Germany).22 

GT patient Osaka-5

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 αIIbβ323 revealed that the amounts of αIIb and β3 in platelets from patient Osaka-5 were 6% and 8% of control platelets, respectively (data not shown). Although the amounts of αIIbβ3 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 cytometry and immunoblot assay

Flow cytometric analysis using various MoAbs and immunoblot assay using rabbit polyclonal antisera specific for αIIbβ3 were performed as previously described.23 24 To examine the expression of αvβ3 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 ELISA

Quantitative enzyme-linked immunosorbent assay (ELISA) was performed to examine the amounts of αvβ3 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 αvβ3 purchased from Chemicon International (Temecula, CA).

Amplification and analysis of platelet RNA

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).

Allele-specific restriction enzyme analysis

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.

Construction of β3 expression vectors

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 αIIbβ3 or αvβ3 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 labeling of the transfected cells

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

Metabolic label with [35S]methionine and pulse chase

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 

Fibrinogen binding assay

Soluble fibrinogen binding assay was performed as previously described.29 For fibrinogen binding to αvβ3, 50 μL aliquots of αvβ3-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 αvβ3. 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 αvβ3 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, αvβ3-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 αIIbβ3, αIIbβ3-transfected cells were examined in the presence or absence of 10 μM FK633 (an αIIbβ3 antagonist) with 1 mM CaCl2 and 10 μg/mL PT25-2 (an αIIbβ3-activating antibody). The following procedures were the same as described above.

Expression level of αIIbβ3 and αvβ3 on platelets from thrombasthenic patient Osaka-5

We examined the surface expression of αIIbβ3 and αvβ3on platelets from patient Osaka-5 using flow cytometry. While the GPIb-specific MoAb AP1 bound equivalently to Osaka-5 and control platelets, the αIIbβ3 complex–specific MoAb AP2, the β3-specific MoAb AP3, and the αIIb-specific MoAb TP80 showed a marked reduction in the expression of αIIbβ3 on Osaka-5 platelets (Figure1A). The amount of αIIbβ3 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 αvβ3 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 αvβ3 expressed on Osaka-5 platelet surface appeared to be 41% of control platelets (Figure 1B). To further examine the expression of αvβ3 in Osaka-5 platelets, we measured the amounts of αvβ3in platelet lysates using sensitive ELISA. The amounts of αvβ3 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 αvβ3 expression in Osaka-5 platelets was about half as much as that in control platelets.

Fig. 1.

Flow cytometric analysis and quantitative ELISA for platelet β3 integrins on control and Osaka-5 platelets.

(A) Expression of αIIbβ3. Washed platelets obtained from a control subject and patient Osaka-5 were incubated with 10 μg/mL AP1 (specific for GPIb), 10 μg/mL AP2 (specific for αIIbβ3 complex), 10 μg/mL AP3 (specific for β3), or 10 μg/mL TP80 (specific for αIIb) for 30 minutes at 22°C. After washing, bound MoAbs were detected by FITC-conjugated goat F(ab′)2 antimouse IgG. MOPC21 (mouse IgG1) was used as a negative control (dotted line). (B) Expression of αvβ3. Control and Osaka-5 platelets were incubated with 5 μg/mL LM609 (specific for αvβ3 complex) for 30 minutes at 22°C. After washing, bound MoAbs were detected by Alexa-conjugated goat F(ab′)2 antimouse IgG. MOPC21 (mouse IgG1) was used as a negative control (dotted line). The amounts of bound LM609 were expressed as MFI from 11 control subjects (mean ± SD) and MFI from patient Osaka-5 (mean of duplicate). (C) The amounts of αvβ3 measured by quantitative ELISA; 100 μL platelet lysate (1 × 106 platelets/μL) was applied to a sandwich ELISA. Standard curve was obtained using purified αvβ3. Data represents the mean ± SD from 11 control subjects.

Fig. 1.

Flow cytometric analysis and quantitative ELISA for platelet β3 integrins on control and Osaka-5 platelets.

(A) Expression of αIIbβ3. Washed platelets obtained from a control subject and patient Osaka-5 were incubated with 10 μg/mL AP1 (specific for GPIb), 10 μg/mL AP2 (specific for αIIbβ3 complex), 10 μg/mL AP3 (specific for β3), or 10 μg/mL TP80 (specific for αIIb) for 30 minutes at 22°C. After washing, bound MoAbs were detected by FITC-conjugated goat F(ab′)2 antimouse IgG. MOPC21 (mouse IgG1) was used as a negative control (dotted line). (B) Expression of αvβ3. Control and Osaka-5 platelets were incubated with 5 μg/mL LM609 (specific for αvβ3 complex) for 30 minutes at 22°C. After washing, bound MoAbs were detected by Alexa-conjugated goat F(ab′)2 antimouse IgG. MOPC21 (mouse IgG1) was used as a negative control (dotted line). The amounts of bound LM609 were expressed as MFI from 11 control subjects (mean ± SD) and MFI from patient Osaka-5 (mean of duplicate). (C) The amounts of αvβ3 measured by quantitative ELISA; 100 μL platelet lysate (1 × 106 platelets/μL) was applied to a sandwich ELISA. Standard curve was obtained using purified αvβ3. Data represents the mean ± SD from 11 control subjects.

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Nucleotide sequence analysis and allele-specific restriction enzyme analysis

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.

Fig. 2.

Analysis of β3 cDNA and theβ3 gene in patient Osaka-5.

(A) Nucleotide sequence analysis of β3 cDNA in patient Osaka-5. The β3 cDNAs from control or Osaka-5 platelets were amplified by RT-PCR. The amplified fragments were directly examined using Taq DyeDeoxy Terminator Cycle Sequencing kit. Samples were run and analyzed on an ABI 373A DNA sequencer. (B) Allele-specific restriction enzyme analysis. The region around exon 5 of the β3 gene was amplified by PCR using primers IIIaE5 and IIIaE6BspHI, followed by digestion with BspHI.BspHI digestion of the PCR products yields 205-bp and 24-bp fragments in normal allele. The A→C substitution abolished a restriction site for BspHI. The resulting fragments were electrophoresed in a 6% polyacrylamide gel; φX174 digested withHaeIII was used as a marker.

Fig. 2.

Analysis of β3 cDNA and theβ3 gene in patient Osaka-5.

(A) Nucleotide sequence analysis of β3 cDNA in patient Osaka-5. The β3 cDNAs from control or Osaka-5 platelets were amplified by RT-PCR. The amplified fragments were directly examined using Taq DyeDeoxy Terminator Cycle Sequencing kit. Samples were run and analyzed on an ABI 373A DNA sequencer. (B) Allele-specific restriction enzyme analysis. The region around exon 5 of the β3 gene was amplified by PCR using primers IIIaE5 and IIIaE6BspHI, followed by digestion with BspHI.BspHI digestion of the PCR products yields 205-bp and 24-bp fragments in normal allele. The A→C substitution abolished a restriction site for BspHI. The resulting fragments were electrophoresed in a 6% polyacrylamide gel; φX174 digested withHaeIII was used as a marker.

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Effect of His280→Pro substitution (Pro280β3) on the expression of αIIbβ3 and αvβ3

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 αIIbβ3 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 αIIbβ3 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 αIIbβ3 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 αvβ3 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 αvβ3 (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 αvβ3 complex (Figure 3D). To rule out the possibility that the normal expression of αvβ3 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 αvβ3 expression. However, the surface expression of αvPro280β3 complex was almost the same as wild-type αvβ3 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 αvβ3 (data not shown). These data indicate that the 887A>C substitution leads to the marked reduction in the amount of αIIbβ3without disturbing αvβ3 expression at least in 293 cells.

Fig. 3.

Effects of His280Pro β3 missense mutation on the expression of αIIbβ3 and αvβ3 in 293 cells.

(A) Flow cytometric analysis of αIIbβ3 on the transfected cell surface. Wild-type or His280Proβ3cDNA was cotransfected into 293 cells with wild-type αIIb cDNA and GFP expression vector pEGFP-C1. The binding of AP2, TP80, and PAC-1 with PT25-2 to the transfected cells was analyzed by flow cytometry 2 days after transfection. Results are representative of at least 3 separate experiments. (B) Immunoprecipitation analysis of biotin surface-labeled transfected cells. The transfected cells were surface-labeled with biotin 2 days after transfection. Immunoprecipitation was then performed using AP3. Precipitates were separated by 6% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. After transfer to a nitrocellulose membrane, precipitated proteins were detected by chemiluminescence. (C) Immunoblot analysis of transfected cells. The transfected cells were lysed and separated by 6% SDS-PAGE under reducing conditions 2 days after transfection. After transfer to a nitrocellulose membrane, αIIb and β3 were detected with a 1:10 000 dilution of rabbit polyclonal anti-αIIbβ3 antibodies. (D) Flow cytometric analysis of αvβ3 on the transfected cell surface. Wild-type or His280Proβ3 cDNA was transfected into 293 cells (i) in the absence or (ii) in the presence of wild-type αv cDNA. The binding of LM609 (specific for αvβ3 complex) or LM142 (specific for αv) to the transfected cells was analyzed by flow cytometry 2 days after transfection. MOPC21 was used as a negative control (dotted line).

Fig. 3.

Effects of His280Pro β3 missense mutation on the expression of αIIbβ3 and αvβ3 in 293 cells.

(A) Flow cytometric analysis of αIIbβ3 on the transfected cell surface. Wild-type or His280Proβ3cDNA was cotransfected into 293 cells with wild-type αIIb cDNA and GFP expression vector pEGFP-C1. The binding of AP2, TP80, and PAC-1 with PT25-2 to the transfected cells was analyzed by flow cytometry 2 days after transfection. Results are representative of at least 3 separate experiments. (B) Immunoprecipitation analysis of biotin surface-labeled transfected cells. The transfected cells were surface-labeled with biotin 2 days after transfection. Immunoprecipitation was then performed using AP3. Precipitates were separated by 6% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. After transfer to a nitrocellulose membrane, precipitated proteins were detected by chemiluminescence. (C) Immunoblot analysis of transfected cells. The transfected cells were lysed and separated by 6% SDS-PAGE under reducing conditions 2 days after transfection. After transfer to a nitrocellulose membrane, αIIb and β3 were detected with a 1:10 000 dilution of rabbit polyclonal anti-αIIbβ3 antibodies. (D) Flow cytometric analysis of αvβ3 on the transfected cell surface. Wild-type or His280Proβ3 cDNA was transfected into 293 cells (i) in the absence or (ii) in the presence of wild-type αv cDNA. The binding of LM609 (specific for αvβ3 complex) or LM142 (specific for αv) to the transfected cells was analyzed by flow cytometry 2 days after transfection. MOPC21 was used as a negative control (dotted line).

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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 αIIbβ3 in the presence of PT25-2 (Figure3A). The PAC-1 binding to αIIbβ3 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).

Effect of missense mutations in β3 on the expression of αIIbβ3 and αvβ3

The different effects of the His280Proβ3 mutation between αIIbβ3 and αvβ3 expression made us further examine the effects of other missense mutations found in GT patients on αvβ3 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 αIIbβ3. Immunoprecipitation of the surface-labeled αIIbβ3-transfected cells using AP3 further showed that the amounts of the mutant αIIbβ3 were markedly reduced compared with wild-type αIIbβ3 (Figure 4B). As shown in Figure 5, each of the Leu117Trp and Cys374Tyr mutations resulted in the marked reduction in αvβ3 expression as well. In sharp contrast, none of Ser162Leu, Arg216Gln, or Arg216Gln/Leu292Ser mutations impaired αvβ3 expression. Of particular interest was the effect of the Arg216Gln/Leu292Serβ3 mutation. Although the Arg216Gln/Leu292Ser mutation completely abolished αIIbβ3 expression, this mutation did not impair αvβ3 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 αvβ3.34 Indeed, the Leu117Trp β3 mutation severely impaired the expression of αv subunits as well as αvβ3, suggesting that the bulk of expressed αv integrins is αvβ3 in these conditions (Figure5).

Fig. 4.

Effects of β3 missense mutations on the expression of αIIbβ3.

(A) Flow cytometric analysis of αIIbβ3on the transfected cell surface. Wild-type β3 cDNA or each mutant β3 was cotransfected into 293 cells with wild-type αIIb cDNA. The binding of AP2 or TP80 to the transfected cells was analyzed by flow cytometry 2 days after transfection. Results were mean ± SD from 3 separate experiments and expressed as percent MFI relative to that of wild-type αIIbβ3. Two-tailed P values for paired samples were obtained by the Student t test (*P < .01, **P < .05). (B) Immunoprecipitation analysis of biotin surface-labeled transfected cells. The transfected cells were surface-labeled with biotin 2 days after transfection. Immunoprecipitation was then performed using AP3. Precipitates were separated by 6% SDS-PAGE under reducing conditions. After transfer to a nitrocellulose membrane, precipitated proteins were detected by chemiluminescence.

Fig. 4.

Effects of β3 missense mutations on the expression of αIIbβ3.

(A) Flow cytometric analysis of αIIbβ3on the transfected cell surface. Wild-type β3 cDNA or each mutant β3 was cotransfected into 293 cells with wild-type αIIb cDNA. The binding of AP2 or TP80 to the transfected cells was analyzed by flow cytometry 2 days after transfection. Results were mean ± SD from 3 separate experiments and expressed as percent MFI relative to that of wild-type αIIbβ3. Two-tailed P values for paired samples were obtained by the Student t test (*P < .01, **P < .05). (B) Immunoprecipitation analysis of biotin surface-labeled transfected cells. The transfected cells were surface-labeled with biotin 2 days after transfection. Immunoprecipitation was then performed using AP3. Precipitates were separated by 6% SDS-PAGE under reducing conditions. After transfer to a nitrocellulose membrane, precipitated proteins were detected by chemiluminescence.

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Fig. 5.

Effects of β3 missense mutations on the expression of αvβ3.

(A) Flow cytometric analysis of αvβ3 on the transfected cell surface. Wild-type β3 cDNA or each mutant β3 was cotransfected into 293 cells with wild-type αv. The binding of LM609 or LM142 to the transfected cells was analyzed by flow cytometry 2 days after transfection. Results were mean ± SD from 3 separate experiments and expressed as percent MFI relative to that of wild-type αvβ3. Two-tailed P values for paired samples were obtained by the Student t test (*P < .01, **P < .05). (B) Immunoprecipitation analysis of biotin surface-labeled transfected cells. The transfected cells were surface-labeled with biotin 2 days after transfection. Immunoprecipitation was then performed using AP3. Precipitates were separated by 6% SDS-PAGE under reducing conditions. After transfer to a nitrocellulose membrane, precipitated proteins were detected by chemiluminescence.

Fig. 5.

Effects of β3 missense mutations on the expression of αvβ3.

(A) Flow cytometric analysis of αvβ3 on the transfected cell surface. Wild-type β3 cDNA or each mutant β3 was cotransfected into 293 cells with wild-type αv. The binding of LM609 or LM142 to the transfected cells was analyzed by flow cytometry 2 days after transfection. Results were mean ± SD from 3 separate experiments and expressed as percent MFI relative to that of wild-type αvβ3. Two-tailed P values for paired samples were obtained by the Student t test (*P < .01, **P < .05). (B) Immunoprecipitation analysis of biotin surface-labeled transfected cells. The transfected cells were surface-labeled with biotin 2 days after transfection. Immunoprecipitation was then performed using AP3. Precipitates were separated by 6% SDS-PAGE under reducing conditions. After transfer to a nitrocellulose membrane, precipitated proteins were detected by chemiluminescence.

Close modal

To elucidate the mechanism of impaired expression of the mutant αIIbβ3, 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 αIIbβ3and αvβ3 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 proαIIbβ3 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 αIIbβ3 was not detectable in Arg216Gln/Leu292Ser mutant, while a small amount of mature αIIbβ3 was observed in the His280Pro mutant. In sharp contrast to αIIbβ3mutants, the kinetics of αvHis280Proβ3 and αvArg216Gln/Leu292Serβ3 biosynthesis were the same as that of wild type, and the normal amount of mature αvβ3 was synthesized at 22 hours after chase (Figure 6B).

Fig. 6.

Pulse chase analysis of β3 integrin biosynthesis.

Wild-type, His280Pro, or Arg216Gln/Leu292Serβ3 cDNA was cotransfected into 293 cells with either (A) wild-type αIIb cDNA or (B) wild-type αv cDNA. Cells were labeled one day after transfection with 0.4 mCi/mL (14.8 MBq) [35S]methionine for 30 minutes and chased with media containing 50 μg/mL of nonradioactive methionine for various periods of time as indicated. Immunoprecipitation was performed using either (A) TP80 or (B) LM142. Precipitates were separated by 6% SDS-PAGE under reducing conditions. Results are representative of 3 separate experiments.

Fig. 6.

Pulse chase analysis of β3 integrin biosynthesis.

Wild-type, His280Pro, or Arg216Gln/Leu292Serβ3 cDNA was cotransfected into 293 cells with either (A) wild-type αIIb cDNA or (B) wild-type αv cDNA. Cells were labeled one day after transfection with 0.4 mCi/mL (14.8 MBq) [35S]methionine for 30 minutes and chased with media containing 50 μg/mL of nonradioactive methionine for various periods of time as indicated. Immunoprecipitation was performed using either (A) TP80 or (B) LM142. Precipitates were separated by 6% SDS-PAGE under reducing conditions. Results are representative of 3 separate experiments.

Close modal

Effect of missense mutations in β3 on the ligand binding function of αIIbβ3 and αvβ3

We then assessed the ligand binding function of the mutant β3 integrins. We measured the binding of FITC-fibrinogen to mutant αIIbβ3 and αvβ3. The αIIbβ3-transfected cells were treated with the αIIbβ3-activating antibody, PT25-2, while αvβ3-transfected cells were treated with 1 mM MnCl2, which induces a high-affinity state of αvβ3. 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 αvβ3. In addition, the blockade of the fibrinogen binding by αIIbβ3-specific antagonist FK633 at 10 μM and αvβ3-specific antagonist cyclo(RGDfV) at 50 μM indicated that the binding was specifically mediated by αIIbβ3 and αvβ3, respectively, in our experimental conditions (Figure 7). Because the expression levels of αIIbβ3 and αvβ3were different in each mutation (Figures 4 and 5), we monitored αIIbβ3 and αvβ3expression by PT25-2 and LM142, respectively, and only analyzed the cells expressing the same levels of αIIbβ3and αvβ3 (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 αIIbβ3 and αvβ3. Ser162Leu mutation markedly impaired the ligand binding to αIIbβ3 but not to αvβ3 at all (Figure 7B). Similarly, Arg216Gln more severely impaired the ligand binding to αIIbβ3 than to αvβ3. Thus, Ser162Leu and Arg216Gln mutations showed a different effect on ligand binding between 2 β3 integrins.

Fig. 7.

Soluble fibrinogen binding to mutant β3integrins.

(A) Dot plots represent FITC-fibrinogen (horizontal) and PT25-2 (vertical) binding to αIIbβ3-transfected cells. The αIIbβ3-transfected cells were treated with 10 μg/mL PT25-2 (an αIIbβ3-activating antibody) for 30 minutes on ice in the presence or absence of 10 μM FK633 (an αIIbβ3 antagonist). After washing, cells were incubated with 150 μg/mL FITC-fibrinogen and phycoerythrin-conjugated antimouse IgG for 25 minutes at room temperature. Then, after 5 minutes incubation with propidium iodine, cells were washed and analyzed by flow cytometry. Because the expression levels of β3 integrins were different in each mutation, we gated and analyzed cells showing the same expression levels of αIIbβ3 for fibrinogen binding. (B) Dot plots represent FITC-fibrinogen (horizontal) and LM142 (vertical) binding to αvβ3-transfected cells. The αvβ3-transfected cells were treated with 1 mM MnCl2 for 30 minutes on ice in the presence or absence of 1 mM RGDW or 50 μM c(RGDfV). LM142 (10 μg/mL) was added simultaneously to the tubes to monitor expression of αvβ3. The following procedures were the same as described above. (C) Fibrinogen binding to αIIbβ3 mutants. Results were mean ± SD from 3 separate experiments and expressed as percent MFI relative to that of wild-type αIIbβ3. Two-tailedP values for paired samples were obtained by the Student t test (*P < .01, **P < .05). (D) Fibrinogen binding to αvβ3 mutants. Results were mean ± SD from 3 separate experiments and expressed as percent MFI relative to that of wild-type αvβ3. Two-tailed Pvalues for paired samples were obtained by the Student ttest (*P < .01, **P < .05).

Fig. 7.

Soluble fibrinogen binding to mutant β3integrins.

(A) Dot plots represent FITC-fibrinogen (horizontal) and PT25-2 (vertical) binding to αIIbβ3-transfected cells. The αIIbβ3-transfected cells were treated with 10 μg/mL PT25-2 (an αIIbβ3-activating antibody) for 30 minutes on ice in the presence or absence of 10 μM FK633 (an αIIbβ3 antagonist). After washing, cells were incubated with 150 μg/mL FITC-fibrinogen and phycoerythrin-conjugated antimouse IgG for 25 minutes at room temperature. Then, after 5 minutes incubation with propidium iodine, cells were washed and analyzed by flow cytometry. Because the expression levels of β3 integrins were different in each mutation, we gated and analyzed cells showing the same expression levels of αIIbβ3 for fibrinogen binding. (B) Dot plots represent FITC-fibrinogen (horizontal) and LM142 (vertical) binding to αvβ3-transfected cells. The αvβ3-transfected cells were treated with 1 mM MnCl2 for 30 minutes on ice in the presence or absence of 1 mM RGDW or 50 μM c(RGDfV). LM142 (10 μg/mL) was added simultaneously to the tubes to monitor expression of αvβ3. The following procedures were the same as described above. (C) Fibrinogen binding to αIIbβ3 mutants. Results were mean ± SD from 3 separate experiments and expressed as percent MFI relative to that of wild-type αIIbβ3. Two-tailedP values for paired samples were obtained by the Student t test (*P < .01, **P < .05). (D) Fibrinogen binding to αvβ3 mutants. Results were mean ± SD from 3 separate experiments and expressed as percent MFI relative to that of wild-type αvβ3. Two-tailed Pvalues for paired samples were obtained by the Student ttest (*P < .01, **P < .05).

Close modal

Among genetic defects responsible for GT phenotype, single amino acid substitutions in each subunit have been especially informative in defining precise structural domains of αIIbβ3 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, αvβ3. In this study we investigated the effects of 6 missense mutations in β3, including His280Pro mutation, on the expression and function of αIIbβ3 and αvβ3in 293 cells. Leu117Trp and Cys374Tyrβ3 mutations impaired both αIIbβ3 and αvβ3 expression, while His280Pro, Ser162Leu, Arg216Gln, and Arg216Gln/Leu292Serβ3 mutations impaired αIIbβ3 expression but not αvβ3 expression. With regard to ligand binding, Ser162Leu and Arg216Gln mutations markedly impaired the ligand binding to αIIbβ3 but not to αvβ3. Our present data demonstrate that some β3 missense mutations have a different impact on the expression and function of αIIbβ3 and αvβ3.

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 αIIbβ3 expression. Except for the Cys374Tyr mutation, His280Pro, Ser162Leu, and Arg216Gln mutations responsible for type II GT phenotype did not impair αvβ3expression in 293 cells, while the Leu117Trp mutation responsible for type I GT phenotype disturbed αvβ3expression. From these data one could argue that different effects of β3 mutations on the biosynthesis of αvβ3 may reflect only the severity of αIIbβ3 deficiency. However, a newly created double mutation, Arg216Gln/Leu292Ser, clearly denied this possibility, because the mutation led to a severe αIIbβ3deficiency but normal αvβ3 expression. These findings strongly suggest that the expression of αIIbβ3 is more strictly regulated than αvβ3.

Fig. 8.

Comparison of integrin β3 amino acid sequences with other integrin β subunits.

The boxed areas are well-conserved residues between several β subunits. Missense mutations examined in this study are also indicated. Leu117 and Cys374 in β3 are well-conserved residues, while Ser162, Arg216, His280, and Leu292 in β3 are rather diverse between 8 β subunits.

Fig. 8.

Comparison of integrin β3 amino acid sequences with other integrin β subunits.

The boxed areas are well-conserved residues between several β subunits. Missense mutations examined in this study are also indicated. Leu117 and Cys374 in β3 are well-conserved residues, while Ser162, Arg216, His280, and Leu292 in β3 are rather diverse between 8 β subunits.

Close modal

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 αIIbβ3(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 proαIIbβ3 was retained and degraded within endoplasmic reticulum.

Because platelets express only a limited number of αvβ3 (about 100 per platelet), we carefully examined the expression levels of αvβ3 in Osaka-5 platelets. In sharp contrast to the markedly impaired αIIbβ3 expression (about 6% of normal), sensitive ELISA as well as flow cytometric analysis showed that Osaka-5 platelets possessed about 50% of the normal αvβ3 content, that is, an apparently higher amount of αvβ3 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 (αIIbβ3, about 6% in Osaka-5 platelets vs about 25% in 293 cells; αvβ3, 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 αvβ3 between human 293 and Chinese hamster ovary cells is likely due to a difference between species.

There are some distinctive features between αIIbβ3 and αvβ3. Treatment of αIIbβ3 with ethylenediaminetetraacetic acid at 37°C dissociates the complex into its individual subunits, while αvβ3 remains a heterodimer.44This difference may reflect tighter cation binding to αvβ3 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 αvβ3 in the presence of Mn2+but not in Ca++, while it binds to αIIbβ3 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 αvβ3 complex–specific MoAb LM609. These findings further indicate structural differences between αIIbβ3 and αvβ3. Employing αv/αIIb chimeras, it has been reported that ligand recognition specificity of αIIbβ3 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, αLβ2 (CD11a/CD18), αMβ2 (CD11b/CD18), and αXβ2 (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 αL, αM, and αX have been found only in combination with β2, biosynthesis of αLβ2, αMβ2, and αXβ2 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 αIIbβ3and αvβ3 expression and function are differently regulated by certain β3 missense mutations. We also suggest that Ser162 and Arg216 residues regulate the stability of αIIbβ3 and αvβ3 differently. These findings would provide insights into the structural requirement for αIIbβ3 and αvβ3function as well as their expression.

We thank Dr Thomas J. Kunicki for the rabbit polyclonal antisera specific for αIIbβ3 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).

Supported in part by grants from the Ministry of Education, Science, and Culture; the Japan Society for the Promotion of Science; and Welfide Medical Research Foundation, Osaka, Japan.

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

1
Hynes
RO
Integrins: versatility, modulation, and signaling in cell adhesion.
Cell.
69
1992
11
25
2
Smyth
S
Joneckis
C
Parise
L
Regulation of vascular integrins.
Blood.
81
1993
2827
2843
3
Shattil
SJ
Function and regulation of the β3 integrins in hemostasis and vascular biology.
Thromb Haemost.
74
1995
149
155
4
Phillips
DR
Charo
IF
Parise
LV
Fitzgerald
LA
The platelet membrane glycoprotein IIb-IIIa complex.
Blood.
71
1988
831
843
5
Brooks
PC
Montgomery
AM
Rosenfeld
M
et al
Integrin αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels.
Cell.
79
1994
1157
1164
6
Liaw
L
Skinner
MP
Raines
EW
et al
The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins: role of αvβ3 in smooth muscle cell migration to osteopontin in vitro.
J Clin Invest.
95
1995
713
724
7
McHugh
KP
Hodivala-Dilke
K
Zheng
MH
Namba
N
Lam
J
Novack
D
Mice lacking β3 integrins are osteosclerotic because of dysfunctional osteoclasts.
J Clin Invest.
105
2000
433
440
8
George
JN
Caen
JP
Nurden
AT
Glanzmann's thrombasthenia: the spectrum of clinical disease.
Blood.
75
1990
1383
1395
9
Coller
BS
Seligsohn
U
Peretz
H
Newman
PJ
Glanzmann thrombasthenia: new insights from an historical perspective.
Semin Hematol.
31
1994
301
311
10
French
DL
Coller
BS
Hematologically important mutations: Glanzmann thrombasthenia.
Blood Cells Mol Dis.
23
1997
39
51
11
Tomiyama
Y
Glanzmann thrombasthenia: integrin αIIbβ3 deficiency.
Int J Hematol.
72
2000
448
454
12
Coller
BS
Cheresh
DA
Asch
E
Seligsohn
U
Platelet vitronectin receptor expression differentiates Iraqi-Jewish from Arab patients with Glanzmann thrombasthenia in Israel.
Blood.
77
1991
75
83
13
Polack
B
Duperray
A
Troesch
A
Berthier
R
Marguerie
G
Biogenesis of the vitronectin receptor in human endothelial cell: evidence that the vitronectin receptor and GPIIb-IIIa are synthesized by a common mechanism.
Blood.
73
1989
1519
1524
14
Duperray
A
Troesch
A
Berthier
R
et al
Biosynthesis and assembly of platelet GPIIb-IIIa in human megakaryocytes: evidence that assembly between pro-GPIIb and GPIIIa is a prerequisite for expression of the complex on the cell surface.
Blood.
74
1989
1603
1611
15
Rosenberg
N
Dardik
R
Rosenthal
E
Zivelin
A
Seligsohn
U
Mutations in the αIIb and β3 genes that cause Glanzmann thrombasthenia can be distinguished by a simple procedure using transformed B-lymphocytes.
Thromb Haemost.
79
1998
244
248
16
Pidard
D
Montgomery
RR
Bennett
JS
Kunicki
TJ
Interaction of AP-2, a monoclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets.
J Biol Chem.
258
1983
12582
12586
17
Newman
PJ
Allen
RW
Kahn
RA
Kunicki
TJ
Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3.
Blood.
65
1985
227
232
18
Shattil
SJ
Hoxie
JA
Brass
LF
Changes in the platelet membrane glycoprotein IIb-IIIa complex during platelet activation.
J Biol Chem.
260
1985
11107
11114
19
Tokuhira
M
Handa
M
Kamata
T
et al
A novel regulatory epitope defined by a murine monoclonal antibody to the platelet GPIIb-IIIa complex (αIIbβ3 integrin).
Thromb Haemost.
76
1996
1038
1046
20
Cheresh
DA
Human endothelial cells synthesize and express an Arg-Gly-Asp-directed adhesion receptor involved in attachment to fibrinogen and von Willebrand factor.
Proc Natl Acad Sci U S A.
84
1987
6471
6475
21
Honda
S
Tomiyama
Y
Aoki
T
et al
Association between ligand-induced conformational changes of integrin αIIbβ3 and αIIbβ3-mediated intracellular Ca2+ signaling.
Blood.
92
1998
3675
3683
22
Pfaff
M
Tangemann
K
Muller
B
et al
Selective recognition of cyclic RGD peptides of NMR defined conformation by αIIbβ3, αvβ3, and α5β1 integrins.
J Biol Chem.
269
1994
20233
20238
23
Tomiyama
Y
Kashiwagi
H
Kosugi
S
et al
Demonstration of a marked reduction in the amount of GPIIb in most type II patients with Glanzmann's thrombasthenia.
Br J Haematol.
87
1994
119
124
24
Tomiyama
Y
Kashiwagi
H
Kosugi
S
et al
Abnormal processing of the glycoprotein IIb transcript due to a nonsense mutation in exon 17 associated with Glanzmann's thrombasthenia.
Thromb Haemost.
73
1995
756
762
25
Tadokoro
S
Tomiyama
Y
Honda
S
et al
A Gln747→Pro substitution in the αIIb subunit is responsible for a moderate αIIbβ3 deficiency in Glanzmann thrombasthenia.
Blood.
92
1998
2750
2758
26
Honda
S
Tomiyama
Y
Shiraga
M
et al
A two-amino acid insertion in the Cys146-Cys167 loop of the αIIb subunit is associated with a variant of Glanzmann thrombasthenia: critical role of Asp163 in ligand binding.
J Clin Invest.
102
1998
1183
1192
27
Zimrin
AB
Eisman
R
Vilaire
G
Schwartz
E
Bennett
JS
Poncz
M
Structure of platelet glycoprotein IIIa: a common subunit for two different membrane receptors.
J Clin Invest.
81
1988
1470
1475
28
Kashiwagi
H
Tomiyama
Y
Honda
S
et al
Molecular basis of CD36 deficiency: evidence that a 478C→T substitution (proline90→serine) in CD36 cDNA accounts for CD36 deficiency.
J Clin Invest.
95
1995
1040
1046
29
Kashiwagi
H
Tomiyama
Y
Tadokoro
S
et al
A mutation in the extracellular cysteine-rich repeat region of the β3 subunit activates integrins αIIbβ3 and αvβ3.
Blood.
93
1999
2559
2568
30
Basani
RB
Brown
DL
Vilaire
G
Bennett
JS
Poncz
M
A Leu117→Trp mutation within the RGD-peptide cross-linking region of β3 results in Glanzmann thrombasthenia by preventing αIIbβ3 export to the platelet surface.
Blood.
90
1997
3082
3088
31
Jackson
DE
White
MM
Jennings
LK
Newman
PJ
A Ser162→Leu mutation within glycoprotein (GP) IIIa (Integrin β3) results in an unstable αIIbβ3 complex that retains partial function in a novel form of type II Glanzmann thrombasthenia.
Thromb Haemost.
80
1998
42
48
32
Newman
PJ
Weyerbusch-Bottum
S
Visentin
GP
Gidwitz
S
White
GC
II
Type II Glanzmann thrombasthenia due to a destabilizing amino acid substitution in platelet membrane glycoprotein IIIa [abstract].
Thromb Haemost.
69
1993
1017
33
Grimaldi
CM
Chen
F
Scudder
LE
Coller
BS
French
DL
A Cys374Tyr homozygous mutation of platelet glycoprotein IIIa (β3) in a Chinese patient with Glanzmann's thrombasthenia.
Blood.
88
1996
1666
1675
34
Honda
S
Tomiyama
Y
Pampori
N
et al
Ligand binding to integrin αvβ3 requires tyrosine 178 in the αv subunit.
Blood.
97
2001
175
182
35
Bodary
SC
McLean
JW
The integrin β1 subunit associates with the vitronectin receptor αv subunit to form a novel vitronectin receptor in a human embryonic kidney cell line.
J Biol Chem.
265
1990
5938
5941
36
Suehiro
K
Gailit
J
Plow
EF
Fibrinogen is a ligand for integrin α5β1 on endothelial cells.
J Biol Chem.
272
1997
5360
5366
37
Loftus
JC
O'Toole
TE
Plow
EF
Glass
A
Frelinger
AL
Ginsberg
MH
A β3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation.
Science.
249
1990
915
918
38
Lanza
F
Stierle
A
Fournier
D
et al
A new variant of Glanzmann's thrombasthenia (Strasbourg I): platelets with functionally defective glycoprotein IIb-IIIa complexes and a glycoprotein IIIa 214Arg→214Trp mutation.
J Clin Invest.
89
1992
1995
2004
39
Fitzgerald
LA
Poncz
M
Steiner
B
Rall
SC
Jr
Bennett
JS
Phillips
DR
Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor α-subunits and platelet glycoprotein IIb.
Biochemistry.
26
1987
8158
8165
40
Moyle
M
Napier
M
McLean
J
Cloning and expression of a divergent integrin subunit β8.
J Biol Chem.
266
1991
19650
19658
41
Ambo
H
Kamata
T
Handa
M
et al
Three novel integrin β3 subunit missense mutations (H280P, C560F, and G579S) in thrombasthenia, including one (H280P) prevalent in Japanese patients.
Biochem Biophys Res Commun.
251
1998
763
768
42
Lee
JO
Rieu
P
Arnaout
MA
Liddington
R
Crystal structure of the A domain from the α subunit of integrin CR3 (CD11b/CD18).
Cell.
80
1995
631
638
43
McKay
BS
Annis
DS
Honda
S
Christie
D
Kunicki
TJ
Molecular requirements for assembly and function of a minimized human integrin αIIbβ3.
J Biol Chem.
271
1996
30544
30547
44
Fitzgerald
LA
Charo
IF
Phillips
DR
Human and bovine endothelial cells synthesize membrane proteins similar to human platelet glycoproteins IIb and IIIa.
J Biol Chem.
260
1985
10893
10896
45
Smith
JW
Piotrowicz
RS
Mathis
D
A mechanism for divalent cation regulation of β3-integrins.
J Biol Chem.
269
1994
960
967
46
Loftus
JC
Halloran
CE
Ginsberg
MH
Feigen
LP
Zablocki
JA
Smith
JW
The amino-terminal one-third of αIIb defines the ligand recognition specificity of integrin αIIbβ3.
J Biol Chem.
271
1996
2033
2039
47
Anderson
DC
Springer
TA
Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins.
Annu Rev Med.
38
1987
175
194
48
Kishimoto
TK
Hollander
N
Roberts
TM
Anderson
DC
Springer
TA
Heterogeneous mutations in the β subunit common to the LFA-1, Mac-1, and p150,95 glycoproteins cause leukocyte adhesion deficiency.
Cell.
50
1987
193
202
49
Etzioni
A
Doerschuk
CM
Harlan
JM
Of man and mouse: leukocyte and endothelial adhesion molecule deficiencies.
Blood.
94
1999
3281
3288
50
Wardlaw
AJ
Hibbs
ML
Stacker
SA
Springer
TA
Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates.
J Exp Med.
172
1990
335
345
51
Arnaout
MA
Dana
N
Gupta
SK
Tenen
DG
Fathallah
DM
Point mutations impairing cell surface expression of the common β subunit (CD18) in a patient with leukocyte adhesion molecule (Leu-CAM) deficiency.
J Clin Invest.
85
1990
977
981

Author notes

Yoshiaki Tomiyama, Dept of Internal Medicine and Molecular Science, Graduate School of Medicine B5, Osaka University, 2-2 Yamadaoka, Suita Osaka 565-0871, Japan; e-mail:yoshi@hp-blood.med.osaka-u.ac.jp.

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