glycoprotein iib-iiia complex classification of gt based on - blood

Molecular Basis for Glanzmann’s Thrombasthenia (GT) in a Compound Heterozygote With Glycoprotein IIb Gene: A Proposal for the Classification of

GT Based on the Biosynthetic Pathway of Glycoprotein IIb-IIIa Complex

By Atushi Kato, Koh Yamamoto, Sumio Miyazaki, Stephanie M. Jung, Masaaki Moroi, and Nobuo Aoki

The genetic basis for Glanzmann’s thrombasthenia (GT) was elucidated on a compound heterozygote with glycoprotein (GP)llb gene: an opal mutation at the end of exon 17 (CGA -+ IGA) results in only a trace amount of GPllb mRNA, and a splicing mutation at the acceptor site of exon 26 (SAG + GAG) causes an in-frame, exon skipping process from exon 25 t o 27. This aberrant transcript encodes a single-chain polypeptide characterized by a 42-amino acid deletion, which in- cludes the proteolytic cleavage site(s) and a unique, proline- rich region at the location corresponding t o the carboxyl- terminal of the normal GPllb archain. These characteristics are shared by a previously reported defective GPllb molecule, which is neither assembled with GPllla nor transported t o the cellular surface. Despite its normal transcription level, expression of the present defective GPllb molecule was

LYCOPROTEIN (GP)IIb-IIIa(aIIb,P3) is a Ca2+- G dependent heterodimer complex that belongs to the integrin famil~, l -~ receptors involved in cell-cell and cell- matrix adhesion. Normal platelets contain approximately 50,000 molecules of GPIIb-IIIa, which comprise 1% to 2% of the total platelet protein.

On platelet activation, GPIIb-IIIa becomes “competent” to bind various RGD sequence-containing ligands such as fibrinogen, von Willebrand factor, collagen, fibronectin, vitronectin, and thrombospondin, thus playing the primary role in thrombus formation at the site of vascular injury.

Hereditary defects of this receptor lead to a bleeding disorder of varying severity, Glanzmann’s thrombasthenia (GT)8,9 As the genetic mode of inheritance is considered to be autosomal recessive in GT, the disease is probably caused by the various combinations of mutations in the GPIIb gene,1° the GPIIIa gene,” or both. Elucidation of the genetic basis for GT is further complicated by the possibility that each mutation of the GPIIb or GPIIIa gene may decrease the expression of another

Recently, we reported the case of an 8-year-old boy with a variant form of GT.17 Western blotting indicated that his

From the First Department of Intemal Medicine, Tokyo Medical and Dental University, Tokyo, Japan; the Department of Pediatrics, Saga Medical School, Saga City, Saga, Japan; and the Department of Protein Biochemistry, Life Science Institute, Kurume University, Ku- rume City, Fukuoka, Japan.

Submitted September 30, 1991; accepted February 14, 1992. Supported by a Grant-in-Aid for Scientific Research on Priority

Areas (1987-1990) from the Ministry of Education, Science and Culture of Japan.

Address reprint requests to Atsushi Kato, MD, The First Depament of Intemal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-lac, Tokyo 113, Japan.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate thk fact.

0 1992 by The American Society of Hematology. 0006-4971 I921 791 2-001 6$3.00/0

significantly decreased (-6% of the control level). Because the precursor GPllb molecule is assembled with GPllla in the endoplasmic reticulum (ER) and its processing, as well as stability, is dependent on the GPllla subunit, the defective GPllb molecule may be rapidly degraded by the intrinsic quality control system of the ER due t o its inability t o form a stable heterodimer complex as a consequence of its misfolded structure. Although we did not confirm that the GPllla genes of this individual were normal, GPllla may be secondarily decreased (-11% of control), because a large part of it could not be complexed, making it vulnerable t o proteolysis. To elucidate the molecular basis for GT, we propose here a classification of GT based on the biosynthetic pathway of the GPllb-llla complex. 8 7992 by The American Society of Hematology.

platelets contained no normal GPIIb, but showed a trace amount of an abnormal GPIIb (-6% with respect to the GPIIb level in a normal individual). This defective molecule had properties similar to those of a previously reported abnormal GPIIb? a single polypeptide chain lacking the cleavage site(s), required for proteolytic processing of the GPIIb precursor to the mature f ~ r m , ’ ~ - ~ ’ with an estimated molecular weight that was approximately 6 Kd less than normal GPIIb in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the nonreduced condition. These observations suggested that the patient was a compound heterozygote with respect to the GPIIb gene, having a silent allele derived from his father and another mutant allele encoding this abnormal molecule from his mot her,

We report here on studies of the molecular basis for the GT of this patient and propose a classification of GT based on the biosynthetic and/or intracellular transport pathway of the GPIIb-IIIa complex.

MATERIALS AND METHODS

Southem blot analysis. DNA used for the following experiments was prepared from the Epstein-Barr virus (EBV)-transformed B cells of the family members. The DNA ( - 3 kg) was digested with 20 to 30 U of each restriction endonuclease under conditions recommended by the suppliers (Takara Shuzo, Osaka, Japan, and Kyoto and Toyobo, Osaka, Japan). Each sample was electrophoresed on a 0.8% agarose gel and then transferred to a Hybond-N membrane (Amersham International, Buckinghamshire, UK). The 2,553-bp fragment of GPIIb cDNA (bases 418 to 2970),19 which was generously provided by Dr J.C. Loftus (Scripps Institute, La Jolla, CA), and the 2,376-bp fragment of GPIIIa cDNA (bases 51 to 2426)u were labeled with [a-32P]dCP using random hexanucleotide primers and Klenow fragment. Hybridization and washing were performed according to the standard conditions suggested by manufacturer.

The patient’s DNA was partially digested by Sau3AI and 15- to 25-kb fragments were collected by sucrose gradient fractionation, ligated to EMBL 3A arms, and packaged using the Gigapack Gold Cloning System (Stratagene, La Jolla, CA). Screening of the library was

Construction and screenirrg of the patient’s genomic library.

3212 Blood, Vol79, No 12 (June 151,1992: pp 3212-3218

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MOLECULAR BASIS AND CLASSIFICATION OF GT 3213

performed using the 32P-labeled GPIIb cDNA probe described above.

Amplification of gemmic DNA fragments by polymerase chain reaction. Genomic DNA fragments containing exon(s) of the GPIIb genelo were amplified by polymerase chain reaction (PCR)’,ul with approximately 1 pg of DNA using each pair of sense (S) and antisense (AS) oligonucleotide primers synthesized by a DNA synthesizer, model 381A (Applied Biosystems, Fostor City, CA) (Fig 1); EX1-5‘PlS: nucleotides (nt) (-)713 -(-)694; EX1-5’P2S: 37-56; EX1-5’P3S: (-)143-(-)124; EX1-5’P4S: (-)318-(-)299; EX1-5’P5S (-)493-(-)474; EX13‘PlAS: 280- 261; EX1-3’P2AS: 94-75; EX1-3’P3AS: (-)80-(-)99, EX1- 3’P4AS: (-)249-( -)268; EX1-3’PSAS: (-)494-( -)513; EX2- 5’PlS: 3609-3628; EX2-3’PlAS: 3833-3814; EX3-5’PlS: 3805- 3824; EX3-3’PlAS: 4019-4000; EX4-5’PlS: 4026-4045; EX4- 3’PlAS: 4270-4251; EX5-5’PlS: 4379-4398; EX5-3’PlAS: 4544- 4525; EX6-5’PlS: 4470-4489; EX6-3’PlAS: 4626-4607; EX7- 5’PlS: 4547-4566; EX7-3’PlAS: 4759-4740; EX8-5’PlS 5045- 5064; EX8-3’PlAS: 5194-5175; EX9-5‘PlS 5228-5247; EX9- 3’PlAS: 5375-5356; EX10-5’PlS: 5440-5459; EXlO-3‘PlAS: 5612- 5593; EX11-5’PlS: 5636-5655; EX11-3’PlAS: 5789-5770; EX12- 5’PlS: 5900-5919; EX12-3’PlAS: 6180-6161; EX13-5’PlS: 8541- 8560; EX13-3’PlAS 8795-8776; EX14-5’PlS 8958-8977; EX14- 3’PlAS: 9095-9076; EX15-5’PlS:9123-9142; EX15-3’PlAS: 9305- 9286, EX16-5’PlS: 9287-9306, EX16-3‘PlAS 9442-9423; EX17- 5‘PlS: 9459-9478; EX17-5’P2S: 9398-9417; EX17-3‘PlAS: 9702- 9683; EX17-3’P2AS: 9742-9723; EX18-5’PlS 9799-9818; EX18- 3’PlAS: 10012-9993; EX19-5’PlS: 10896-10915; EX19-3‘PlAS: 11054-11035; EX20-5’PlS: 11098-11117; EX20-3’PlAS: 11316- 11297; EX21-5’PlS: 11834-11853; EX21-3’PlAS: 11998-11979; EX22-5 ‘P 1 S: 12688- 12669; EX23- 5’PlS: 13234-13253; EX23-3’PlAS: 13383-13364; EX24-5‘PlS: 13438-13457; EX24-3’PlAS: 13623-13604; EX25-5’PlS 13636- 13656; EX25-RlS: 13750-13769; EX25-WS: 13787-13806; EX25- 3’PlAS: 13858-13839; EX26-5’PlS: 13905-13924; EX26-5’P2S: 13870-13889; EX26-3’PlAS: 14125-14106; EX26-3IP2AS: 14007- 13988; EX27-5’PlS: 14486-14505; EX27-R2AS: 14581-14562; EX27- RlAS: 14594-14575; EX27-3‘PlAS: 14715-14696; EX28-5’PlS: 14863-14882; EX28-3’PlAS: 15034-15015, EX29-5’PlS: 15140- 15159; EX29-3’PlAS: 15359-15340, EX30-5’PlS: 16915-16934; EX30-3’PlAS: 17282-17263.

12536- 12555; EX22-3 ’P1 AS:

PCR amplification was performed in an automatic thermocycler (Zymoreactor; Taiyo, Tokyo, Japan) for 35 cycles at 94°C (1 minute), 50 to 60°C (1 minute), and 74°C (1.5 minutes), followed by a 5-minute extension at the end. One tenth of the PCR products were analyzed by electrophoresis on a 0.8% to 5% agarose gel. For the sequencing reaction, template fragments were purified with a Geneclean kit (BIO 101, La Jolla, CA).

Of the five positive clones derived from the patient’s genomic DNA library, template DNA fragments were subcloned into pUC19 and then the nucleotides were sequenced with a Sequanase kit (US Biochemical, Cleveland, OH) using [a-3SS]dCTF’ and primers supplied by the manufacturer or the synthetic oligonucleotide primers described above. Nucleotides on

Nucleotide sequencing.

0 ’ 9 P t M k b

EXONS 1 10 17 26 30

Fig 1. Sequence analysis of the PCR-amplified genomic DNA fragments of the patient’s GPllb gene. Solid boxes represent exons.10 Broken lines {below) indicate the nucleotide regions amplified by PCR and sequenced. The positions of two point mutations {open and closed circles) in the patient’s GPllb gene are indicated.

both strands were sequenced. Nucleotides of the PCR-amplified fragments were sequenced either after subcloning into pUC19 or directly using one oligonucleotide primer with a single-stranded template DNA amplified by another primer.az6

cDNA-PCR using platelet RNA was performed under the previously described condition^.*^-^^ Briefly, cDNA was synthesized with approximately 50 ng of total platelet RNA using retroviral reverse transcriptase, RAV-2 (Takara Shuzo, Kyoto, Japan), and oligo(dT) or random hexanucleotide primers. Then, two consecutive PCRs were performed using nested primers: with EX25-R1S and EX27-R1AS or oligo(dT) for the first PCR, and then with EX25-R2S and EX27-R2AS for the second PCR.

The RNase protection assay was performed essentially as described.30 The 394-bp SacI-StuI GPIIb cDNA fragment was ligated into the Sad-SmaI site of the pSPT18 vector (Boehringer- Mannheim, Germany). After cleavage with EcoRI, an antisense RNA probe was synthesized with SP6 RNA polymerase (Boeh- ringer-Mannheim) and [a-32P]Cr‘P. Approximately 50 ng of platelet RNA was hybridized overnight with 5 x lo6 cpm of the labeled probe, followed by digestion with RNase A and T1, and then electrophoresed on a 5% polyacrylamide gel containing urea.

After autoradiography, densitometric analysis was performed using a Digital Densitorol DMU-33C (Advantec Tokyo, Tokyo, Japan).

Analysk ofplatelet “A.

RESULTS

Analysis of the GPIIb and IZIa genes. To elucidate the genetic defects of the patient, Southern blot analysis was performed with the patient’s DNA using GPIIb or GPIIIa cDNA probes. However, after digestion of the patient’s DNA by the following restriction enzymes: BamHI, Bgl 11, Dra I, EcoRI, EcoRV, HindIII, Hpa I, K j n I, Pst I, Sac I, Stu I, Xba I, and Xho I, neither of the probes detected any differences from control DNA (data not shown). Since it was suggested from Western blot analysis that the patient was a compound heterozygote of the GPIIb gene,17 the nucleotide sequences of all 30 exons and the exon-intron boundaries of the GPIIb genelo were determined. The results showed two independent mutations: an opal mutation at the end of exon 17 C G A --* TGA, Arg 584 + stop) (Fig 2A) and another one abolishing the consensus splice acceptor site ( -3)31,32 of exon 26 (GAG += G AG) (Fig 2B).

To determine the origin of these twomutant alleles, exons 17 and 26 with their flanking regions were amplified by PCR, followed by digestion with Tag I or Mva I, as the restriction sites of these enzymes were abolished by each of the mutations, respectively. The agarose gel electrophoresis indicated that the opal mutation was derived from the patient’s father (Fig 3A) and the splicing mutation from his mother (Fig 3B). These results were concordant with the phenotypic analysis by Western blotting of the whole platelet protein of the family members.

cDNA-PCR with platelet RNA and the nucleotide sequence of aberrant cDNA. Previous studies on various hereditary disorders and the in vitro mutagenesis model indicate that a nonsense mutation often decreases the mRNA leve126,33-35 and a mutation in the splice acceptor site leads to either activation of the cryptic acceptor sites or an exon skipping p r o c e ~ s . ~ ~ - ~ ~

To determine which splicing pattern works in this case,




3214

A KAT0 ET AL

Normal Mutant

Phe 582

Leu 583

T A C G T

C T T T T

EXON D-- 17 Arg584 [ G

4 A - 4

INTRON A'

__o G I! A

STOP

Normal Mutant

Fig 2. Direct sequencing of the nucleotides encompassing exons 17 and 26. Exons 17 and 26 with flanking regions were amplified by PCR with approximately 1 pg of the patient's DNA and oligonucleotide primer pairs of either EX17-5'PlS and EX17-3'PlAS. or EX26-5'PlS and EX26-3'PlAS. respectively. Nucleotides were sequenced directly as described in the Materials and Methods. The base changed by each mutation is indicated by a letter in a box (right side): (A) CGA + I G A at the end of exon 17, and (E) GAG + GAG at the splice acceptor site (-3) of exon 26. Presence of both the normal and mutated bases was confirmed by sequencing nucleotides of the subcloned fragments of PCR-amplified regions and of five positive clones from the patient's genomic library.

cDNA was synthesized on the total RNA extracted from the platelets of the patient and his mother. Then, two consecutive PCRs were performed using nested primers encompassing exon 25 through exon 27. The results indicated the presence of mRNA of two different sizes in thcir platelets: one is the normally spliced, 202-nt mRNA (exon 25-26-27), and the other is a shorter mRNA of 76-nt (Fig 4).

It was conceivable from its size and the restriction enzyme analysis (data not shown) that the latter aberrant

A Taq I 7

TCGA 1 T

-b

1 EXON- c

Normal -1 185 bp 59 bp

Mutant I 1 244 bp

C P F M

mRNA was generated through exon skipping; the donor site of exon 25 was joined to the acceptor site of exon 27. To confirm this putative, exon skipping process, nucleotides of this aberrant cDNA were sequenced. The results proved that the whole nucleotide sequence of exon 26 was deleted and indicated that the 3' terminal base of exon 25 was directly joined to the 5' terminal base of exon 27 (Fig 5).

RNase protection assay. To estimate the amount of mRNA transcribed from these two mutant alleles, an

B Mva I

7 CCAGG 1 G

-b

I EXON 26 1 c

Normal U-1 27bp 187bp

Mutant 0 1 214bp

C P F M

Fig 3. Agarose gel electrophoresis of PCR products of exons 17 and 26, followed by digestion with Taq I (exon 17) and Mva I (exon 26). respectively. PCR was performed using the same primer sets described in Fig 2. (A) Taq I digestion of exon 17 with flanking regions yields 185-bp and 59-bp fragments in the normal allele, but only a 244-bp fragment is observed in the mutant allele. (E) Mva I digestion of exon 26 with flanking regions yields 187-bp and 27-bp fragments in the normal allele, but only a 214-bp fragment is observed in the mutant allele. C, P, F, and M denote DNA from the control, the patient, the patient'sfather, and the patient's mother, respectively. The 59-bp and 27-bp fragments are not shown. Size markers are de- picted on the right.





Fig 4. cDNA-PCR from platelet RNA. After cDNA synthesis, half of the reaction mixture was subjected t o PCR using nested primers as described in the Mate- rials and Methods. One tenth of the final products was electrophoresed on a 2% agarose gel. C, P, and M denote platelet RNA of the control, the patient, and the patient's mother, respectively. The 202-bp fragment represents the cDNA of normally spliced, exon 25-26-27 mRNA, and the 76-bpfragment (arrow) observed in lanes P and M is derived from the mutant mRNA, where exon 26 is skipped. The relative amounts of these two cDNAfrag- ments were not constant and either one of them was amplified in the other tubes under the same conditions (data not shown). Size markers are on the right.

RNase protection assay was RNA (Fig 6).

EX25- R 1 S -+ + EX27-R 1AS EXON 25 ]-<-I EXON26 4-

EX25- R2S + + EX27-R2AS

b Normal 202 bp

Mutant 76 bp

C P M -291 1-21 0 1-1 62

-79

performed on the platelet

After RNase digestion, the normally spliced, 394-nt fragment, as well as 151- and 117-nt fragments derived from the aberrant mRNA, in which exon 26 was skipped, were

A C G T

EXON 25

EXON 27

,c1

\;J Fig 5. Nucleotide sequence of the aberrant cDNA. The 76-bp cDNA

fragment in lane P of Fig 4 was subcloned into pUC 19, and then the nucleotides were sequenced. The 3' end of exon 25 is directly joined to the 5' end of exon 27.

protected in the patient's mother's platelets (Fig 6, lane 4). Densitometric analysis of these fragments indicated that the overall amount of the aberrant mRNA was comparable to that of its normal counterpart, after correction for the difference in length (data not shown). Although both the abnormal 151- and 117-nt fragments in the patient's platelets exhibited even higher density than those of the mother's, 394-nt fragment could not be observed (Fig 6, lane 3). These results indicate that in the patient's platelets, mRNA transcribed from the allele with an opal mutation was significantly decreased and at most below the level detect- able by the RNase protection assay.

DISCUSSION

We analyzed the genetic basis for GT in a family and found two independent mutations in the GPIIb gene. An opal mutation results in the significant decrease in mRNA. A splicing mutation causing an in-frame exon skip results in a normal amount of the aberrant mRNA encoding a truncated GPIIb molecule with a 42-amino acid deletion.

Nonsense mutations have been reported to decrease mRNA levels of various mammalian genes?h.33-3s However, it is controversial whether the primary causative defect resides in the intranuclear metabolism, nuclear-cytoplasmic transport, or the cytoplasmic instability of mRNA.33-35

The deleted 42-amino acid region contains the proteolytic cleavage site(s) required for conversion of the GPIIb precursor to the mature form.1y-2' It is part of the unique carboxyl-terminal region of the a-chain of GPIIb'9.2' and is abundant in proline residue^'^ that prevent a-helix formation. Therefore, the exon skipping process from exon 25 to




3216 KAT0 ET AL

1 2 3 4 5

151-

117-

inability to form a stable heterodimer complex as a consequence of its misfolded structure.

On the other hand, the amount of GPIIIa, which is indistinguishable from the control GPIIIa by Western blot analysis, is also significantly decreased ( - 11% of con-

Although it has not been confirmed whether the GPIIIa genes of the patient are normal, most of GPIIIa may be secondarily decreased without forming the heterodimer complex, because only a defective GPIIb molecule is present.

Before discovery of the GPIIb-IIIa abnormality in GT, Caen et al proposed the classification based on the platelet fibrinogen content and clot r e t r a c t i ~ n . ~ . ~ ~ Recently, George et al classified GT based on the platelet GPIIb-IIIa content: type I (<5% of normal), type I1 (10% to 20% of normal), and variants (half-normal to normal amount of functionally defective molecules).’ In addition, a classification into + and 0 variants according to the presence or absence of GPIIb-IIIa complex on the platelet surface has been pr0posed.4~ Although classification of GT based on the amount of platelet GPIIb-IIIa is convenient, it does not accurately reflect the functional abnormality on the molecular level.

At present, only seven different types of mutations in the GPIIb or IIIa gene, including two types of the present case, have been characterized in GT patients.12.sn-s3 However, for a concise correlation between mutations in these genes and molecular defects leading to the thrombasthenic pheno- tvDes. another classification of GT mav be useful. In other

25 26 27 28 I trol).I7

25 D

27 28

Fig 6. RNase protection assay on platelet RNA. Platelet RNA was hybridized to a “P-labeled antisense RNA probe corresponding to nt 2845 to 2452 of GPllb cDNA. After RNase digestion, the samples were electrophoresed. Lane 1: probe only; lanes 2, 3, 4: RNA from the control, the patient, and the patient‘s mother, respectively; lane 5: yeast tRNA. Lines on the left indicate the positions of the 440-nt fragment of the probe, normal protected fragment of 394 nt (exon 25 through 28). and two smaller fragments of 151 nt (exon 25) and 117 nt (exon 27-28) derived from the aberrant mRNA.

27 results in a single-chain polypeptide structurally quite distinct from the normal precursor molecule.

We have reported another GT patient with a variant GPIIb, which was not associated with GPIIIa or expressed at the platelet surface.IR The defective GPIIb of the present case is indistinguishable from this GPIIb, as evidenced by Western blot ana1y~is.I~

During the early stage of biosynthesis, precursor GPIIb is assembled with GPIIIa in the endoplasmic reticulum (ER) and then transported to the Golgi complex.13J4 For the expression of these glycoproteins on the cell surface, the formation of a complex between GPIIb and GPIIIa is requisite.13--’6

In other words, excess chains, either as free components or as partially assembled complexes, may be rapidly degraded as observed with the T-cell antigen receptofl or acetylcholine receptor!’ Taken together, the defective GPIIb of the present case may be rapidly degraded by the intrinsic quality control system in the ER,4247 due to its

,. ,

words, GT can be classified in terms of the biosynthetic stages of the GPIIb-IIIa complex predominantly impaired by mutation(s) in the GPIIb gene or GPIIIa gene. In essence, the classification is based on whether these two molecules have been assembled to form a stable heterodimer complex (postassembly stage) or not (preassembly stage).

The rationale for this classification is that the bleeding tendency in GT seems to be largely dependent on the amount of the “functional” GPIIb-IIIa complex on the platelet surface, and for expression of the complex, assembly between pro-GPIIb and IIIa in the ER is prerequisite.I3-I6 If a mutation in GPIIb or IIIa gene does not prohibit heterodimer formation, a large portion of even an aberrant complex would be expressed on the platelet surface; such a defect can be classified as a mutation impairing predominantly the postassembly stage.

In contrast, any mutation that causes misfolding of the molecule itself or prevents assembly would significantly decrease the amount of both molecules; such a defect can be classified as a mutation impairing predominantly the preassembly stage. Misfolded molecule may be sorted to the degradation pathway either after binding to such luminal ER proteins as Bip (binding protein) or by forming insoluble agg1-egates.4~~’

A GPIIb gene deletion (-4.5 kb) reported by Burk et al,I2 as well as two types of deletion in the GPIIb or IIIa gene reported by Ncwman et a1,52 may belong to this





category. In general, most of the so-called type I and type I1 GTs9,5“-57 seem to belong to this category.

These preassembly stage defects may be subdivided into two categories on the mRNA level, pretranslation and posttranslation defects, where the decrease of each subunit is attributed to either a decreased level of mRNA or an increased degradation of the unstable molecule despite the presence of a normal amount of mRNA, respectively. From this point of view, the opal mutation in exon 17 of the present case belongs to the pretranslation defect, and the splicing mutation of exon 26, to the posttranslation defect. A large insertion ( - 7 kb) in the GPIIIa gene reported by Bray and Shumanso may also belong to the pretranslation defect.

In the postassembly category of mutations, defects in the receptor function of the complex, rather than its quantity, are the major determinant of the severity of the bleeding tendency. Most of the “variant” types,51,58-62 including the

point mutation in the GPIIIa gene reported by Loftus e t a151,58 and some of the “type 11” GTs, may belong to this postassembly stage.

Thus the classification proposed should more concisely describe the molecular basis for each case of GT. To begin to categorize the thrombasthenic phenotype for further molecular studies, the platelet flow cytometry using monoclonal antibodies for GPIIb-IIIa63.64 may be useful. Precise analyses of molecular defects caused by various mutations will elucidate the biological importance of each structural domain of GPIIb-IIIa in maturation, the intracellular sorting process, and ligand-binding activity.

ACKNOWLEDGMENT

The authors thank Dr K. Yamamoto for supplying the EBV stock and help in establishing cell lines, Dr S. Hirosawa for useful discussions, and H. Maeda for typing the manuscript.

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SUPPI 1)




1992 79: 3212-3218

A Kato, K Yamamoto, S Miyazaki, SM Jung, M Moroi and N Aoki glycoprotein IIb-IIIa complexclassification of GT based on the biosynthetic pathway ofheterozygote with glycoprotein IIb gene: a proposal for the Molecular basis for Glanzmann's thrombasthenia (GT) in a compound

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