Molecular Genetic Analysis of a Compound Heterozygote for the Glycoprotein (GP) IIb Gene Associated With Glanzmann’s Thrombasthenia: Disruption of the 674-687 Disulfide Bridge in GPIIb Prevents Surface Exposure of GPIIb-IIIa Complexes

Abstract

This work was aimed at elucidating the molecular genetic lesion(s) responsible for the thrombasthenic phenotype of a patient whose low platelet content of glycoprotein (GP) IIb-IIIa indicated that it was a case of type II Glanzmann’s thrombasthenia (GT). The parents did not admit consanguinity and showed a reduced platelet content of GPIIb-IIIa. Polymerase chain reaction (PCR)–single-stranded conformational polymorphism analysis of genomic DNA showed no mutations in the patient’s GPIIIa and two novel mutations in the GPIIb gene: one of them was a heterozygous splice junction mutation, a C→A transversion, at position +2 of the exon 5-intron 5 boundary [IVS5(+2)C→A] inherited from the father. The predicted effect of this mutation, insertion of intron 5 (76 bp) into the GPIIb-mRNA, was confirmed by reverse transcription-PCR analysis of platelet mRNA. The almost complete absence of this mutated form of GPIIb-mRNA suggests that it is very unstable. Virtually all of the proband’s GPIIb-mRNA was accounted for by the allele inherited from the mother showing a T2113→C transition that changes Cys674→Arg674 disrupting the 674-687 intramolecular disulfide bridge. The proband showed a platelet accumulation of proGPIIb and minute amounts of GPIIb and GPIIIa. Moreover, transfection and immunoprecipitation analysis demonstrated that [Arg674]GPIIb is capable of forming a heterodimer complex with GPIIIa, but the rate of subunit maturation and the surface exposure of GPIIb-IIIa are strongly reduced. Thus, the intramolecular 674-687 disulfide bridge in GPIIb is essential for the normal processing of GPIIb-IIIa complexes. The additive effect of these two GPIIb mutations provides the molecular basis for the thrombasthenic phenotype of the proband.

A 1063G→A mutation in exon 12 of glycoprotein (GP)IIb associated with a thrombasthenic phenotype: mutation analysis of [324E]GPIIb

“…The heterozygous relatives of the proband showed a reduced platelet expression of GPIIb‐IIIa. This finding is in line with observations made in heterozygous states for other GPIIb mutations (Ferrer et al , 1996; González‐Manchón et al , 1999; Tao et al, 2000). The reason for this reduction has not yet been elucidated.…”

“…GPIIb is synthesized as a single peptide (proGPIIb) that undergoes enzymatic cleavage in the Golgi apparatus to yield the heavy (GPIIbH) and light (GPIIbL) chains. Previous experimental evidence indicates that proGPIIb requires its association with GPIIIa to migrate from the endoplasmic reticulum into the Golgi (Duperray et al , 1987, 1989; González‐Manchón et al , 1999). Thus, a lack of GPIIbH in cells producing proGPIIb would indicate the inability of proGPIIb to complex with GPIIIa.…”

“…Labelling and immunoprecipitation analysis of total GPIIb‐IIIa complexes from transfected CHO cells Biotinylation and immunoprecipitation analysis of total GPIIb/IIIa in CHO cells co‐expressing GPIIIa and either normal or mutant GPIIb were performed as previously described (González‐Manchón et al , 1999). Briefly, transfected cells were treated with lysis buffer [50 mmol/l Tris, pH 7·4, 150 mmol/l NaCl, 2 mmol/l phenylmethyl sulphonyl fluoride (PMSF), 1% Triton X‐100, 0·05% Tween 20 and 0·03% sodium azide] for 30 min at 4°C and then incubated in PBS containing 5 mmol/l D‐biotin‐N‐hydroxy succinimidester (biotin‐NHS; Boehringer Mannheim) for 1 h at room temperature.…”

Singlet Oxygen Inhibits Agonist-Induced P-Selectin Expression and Formation of Platelet Aggregates

“…I In proteins, methionine or cysteine is the main physiologic reaction partners of singlet ~~y~~n; also unsaturated fatty acids or cholesterol could interact with singlet oxygen (18,19). Therefore, although the exact reaction receptor for singlet oxygen in platelets remains to be established, possible candidates could be singlet oxygensusceptible, functionally important proteins such as GP Mb/ma (20), fibrinogen, von Willebrand factor (8), or enzymes or intermediates of the prostaglandin me-t~6c~Iis~n of platelets (21)(22)(23).…”

Expanding the Mutation Spectrum Affecting αIIbβ3 Integrin in Glanzmann Thrombasthenia: Screening of theITGA2BandITGB3Genes in a Large International Cohort

“…pro-αIIbβ3 maturation [Gonzalez-Manchon et al, 1999]. Molecular modeling is illustrated in Figure 3 for the novel p.Gly823Glu (G792E), p.Leu955Gln (L924Q), and p.Thr984Lys (T953K) mutations located in GT27, GT21, and GT72, respectively.…”